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Mol Cell Biol, May 1998, p. 3034-3043, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutation in the Arabidopsis PASTICCINO1
Gene, Which Encodes a New FK506-Binding Protein-Like Protein, Has a
Dramatic Effect on Plant Development
Paola
Vittorioso,
Rachel
Cowling,
Jean-Denis
Faure,
Michel
Caboche, and
Catherine
Bellini*
Laboratoire de Biologie Cellulaire,
INRA-Centre de Versailles, 78026 Versailles Cedex, France
Received 22 August 1997/Returned for modification 2 October
1997/Accepted 16 February 1998
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ABSTRACT |
The pasticcino (pas) mutants of
Arabidopsis thaliana are a new class of plant developmental
mutants; members of this class show ectopic cell proliferation in
cotyledons, extra layers of cells in the hypocotyl, and an abnormal
apical meristem. This phenotype is correlated with both cell division
and cell elongation defects. There are three complementation groups of
pas mutants (pas1, pas2, and
pas3, with, respectively 2, 1, and 4 alleles). Here we
describe in more detail the pas1-1 allele, which was
obtained by insertional mutagenesis. The PAS1 gene has been
cloned and characterized; it encodes an immunophilin-like protein
similar to the p59 FK506-binding protein (FKBP52). PAS1 is
characterized by an FKBP-like domain and three tetratricopeptide repeat
units. Although the presence of immunophilins in plants has already
been demonstrated, the pas1-1 mutant represents the first
inactivation of an FKBP-like gene in plants. PAS1
expression is altered in pas1 mutants and in the
pas2 and pas3 mutants. The expression of the
PAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability
to stimulate cell division. These results are of particular relevance
as they show for the first time that an FKBP-like protein plays an
important role in the control of plant development.
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INTRODUCTION |
In flowering plants, morphogenesis
depends on the control of the pattern and numbers of cell divisions and
on the control of cell elongation. Although there are many examples of
controlled patterns of cell division, we still know very little about
how local patterns of cell division are established and maintained (30). In Arabidopsis thaliana, the roles of cell
division control in the development of the embryo, the shoot, and the
root have been extensively studied (reviewed in references
29 and 30). In the last few
years, much progress has been made in this field by the isolation of
mutants in which single-gene mutations affect specific modes of cell
division control. Some of the corresponding genes have been cloned from
A. thaliana (SHOOT MERISTEMLESS
[STM] and SCARECROW [SCR]) maize
(KNOTTED1), and petunia (NO APICAL MERISTEM)
(reviewed in reference 30). These genes do not seem to specify components of the cell division machinery, but they are
thought to act upstream in the control of cell division. The elements
at the interface between genes like STM and SCR
and cell cycle regulators, such as cyclins and the CDC
genes, are still unknown.
The growth and differentiation of higher plants is also greatly
dependent on environmental stimuli, such as light and temperature, and
on endogenous factors, such as phytohormones. Cytokinins (CKs) were
originally discovered because of their ability to promote, along with
auxins, plant cell division and organogenesis (reviewed in reference
9). Although this discovery initiated a vast amount of fundamental and applied research on the hormonal control of cell
proliferation and regeneration, the mechanisms by which auxins and CKs
act and interact at the molecular level are unknown. Steroid-like plant
growth factors termed brassinosteroids (BR) were first characterized as
inducing cell elongation in synergy with auxin, but recently these
hormones have also been found to control plant cell divisions and
morphogenesis (15; reviewed in reference
11).
The genetic and molecular analysis of hormonal mutants is proving to be
a powerful tool for unraveling the mode of action of these molecules.
In an attempt to understand the mode of action of CKs and their
molecular relationships with auxins in promoting plant cell division,
we looked for Arabidopsis mutants with phenotypes which were
affected by exogenously applied CKs. We have previously reported the
isolation of the pasticcino mutants (pas1,
pas2, and pas3) which are affected in both
embryonic and vegetative development. Their phenotypes are similar to
that of wild-type shoots which have been regenerated in vitro from
explants, in the presence of an unbalanced auxin/CK ratio in the medium
(12).
The pas1-1 mutant was isolated from the transfer DNA (T-DNA)
mutant collection of INRA-Centre de Versailles (2, 12). Here
we describe the cloning of the PAS1 gene from the
T-DNA-tagged pas1-1 allele. PAS1 codes for an
immunophilin-like protein similar to the FK506-binding proteins (FKBP).
We also demonstrate that the PAS1 mRNA steady-state level is
increased in the presence of CK and that PAS1 gene
expression is affected in the other pas mutants.
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MATERIALS AND METHODS |
Arabidopsis lines and growth conditions.
Seeds
from A. thaliana Heynh, ecotype Columbia (Col0) and ecotype
Wassilewskija (WS), were kindly provided by J. Giraudat (CNRS, Gif sur
Yvette, France) and by K. Feldman (University of Arizona, Tucson,
Ariz.), respectively. The mutagenized lines were produced as already
described (2, 12). For growth in the greenhouse, seeds were
sown on soil and seedlings were transferred into individual pots 10 days after germination. Plants were grown under the following
conditions: 16 h of light, 20 to 25°C day temperature, and 10 to
15°C night temperature. For in vitro growth, seeds were sterilized
and grown as already described (12).
Benzyladenine (BA), zeatin, 1-(2-chloropyrid-4-yl)-3-phenylurea, and
picloram, an auxin analog, were filter sterilized and added to the
medium at increasing concentrations from 0 to 10 µM.
Cytological analysis.
For light microscopy, seedlings were
fixed in 4% formaldehyde-0.2% glutaraldehyde and then embedded in
Historesin (Leica, Rueil Malmaison, France) in accordance with the
manufacturer's instructions. Semithin sections (3 to 5 µm thick)
were cut on a Jung RM microtome, stained with 0.05% methylene blue,
and examined with a Nikon Microphot FXA microscope.
Isolation of the pas1-1 mutant.
The
pasticcino1-1 (pas1-1) mutant was identified in
the progeny of T-DNA-mutagenized Arabidopsis ecotype WS
lines produced at the Station de Génétique et
Amélioration des Plantes (Versailles, France) and had been
previously screened for resistance to the Basta herbicide (2,
12). Screening for mutants was based on the early phenotype of
9-day-old plants grown both in the light and in the dark. It was
determined that pas1-1 contained a single T-DNA insert, and
linkage of the T-DNA insert to the PAS1 gene was tested.
More then 100 putative heterozygotes (kanamycin-resistant seedlings
with a wild-type phenotype) each produced 25% mutant progeny. Plants
heterozygous for the mutation were self-fertilized, and the
transmission of the phenotype was confirmed in the M3 generation.
Isolation of genomic DNA and cDNA PAS1 clones.
Seeds harvested from pas1-1 heterozygous plants were grown
in vitro as stated above. Fourteen-day-old pas1-1 mutants
were harvested for DNA extraction. The BamHI and
XbaI genomic DNA fragments adjacent to the right border of
the single T-DNA insert were isolated by the strategy of kanamycin
rescue (6). pas1-1 genomic DNA was double
digested with PstI-BamHI and
PstI-XbaI and ligated, respectively, into
PstI-BamHI- and
PstI-XbaI-digested pResc38 vectors.
Transformation of high-efficiency Escherichia coli DH12S competent cells (1010 CFU/µg of DNA) was performed by
electroporation, and recombinant clones were checked both by PCR
analysis and Southern blot hybridization.
Genomic DNA flanking the left end of the T-DNA insert was isolated by
inverse PCR. DNA from homozygous
pas1-1 mutants was
digested
with
XbaI and then ligated for 16 h at 16°C in a
200-µl
reaction mixture. The ligation mixture was extracted with
phenol-CHCl
3 and with CHCl
3, and DNA was
precipitated with ethanol. The DNA
was then added to a PCR mixture.
Synthetic oligonucleotides corresponding
to the left border of the
T-DNA were used as primers. The PCR
product was digested with
XbaI and
HindIII restriction enzymes
and then
cloned into the pBluescript SK+ vector (Stratagene).
Genomic DNA flanking both the right and left borders of the T-DNA
insert was used to screen an
Arabidopsis Columbia genomic
library (EEC-BRIDGE
Arabidopsis DNA Stock Center, Cologne,
Germany)
and an
Arabidopsis cDNA library (
31).
NotI cDNA inserts were
cloned into the pBluescript SK+
vector (Stratagene) for restriction
analysis, and subclones were used
for sequencing. DNA sequencing
was performed by using
Taq
DNA polymerase, dye primers, and an
ABI 373A automated DNA sequencer as
recommended by the manufacturer
(Applied Biosystems). Standard
molecular techniques were used
throughout (
36). The analysis
of
PAS1 cDNAs and derived protein
sequences was performed in
part with the GCG and BLAST computer
programs and in part by the
National Center for Biotechnology
Information, Bethesda, Md. RNA from
pas1-2 seedlings was reverse
transcribed with a poly(dT)
primer. Specific primers were used
to amplify the
pas1-2
cDNA sequence, which was cloned into pBluescript
SK+. Clones from
several independent PCRs on independent cDNA
samples were sequenced.
Southern and Northern blot analysis.
Arabidopsis
genomic DNA was isolated as previously described (6).
Southern blots on Hybond N membranes were produced as described by the
manufacturer (Amersham).
For RNA extraction, 9-day-old wild types (WS and Col0) and
pas mutants were harvested and immediately frozen in liquid
nitrogen.
Total RNA was isolated by grinding the tissues in liquid
nitrogen.
The samples were then vortexed for 3 min in the presence of
an
extraction buffer (0.1 M LiCl, 0.1 M Tris-HCl [pH 8], 0.01 M EDTA,
1% sodium dodecyl sulfate-phenol-chloroform mixture (1:1:1). Several
phenol-chloroform extractions were then performed. RNA was precipitated
overnight at 4°C with 1 volume of 4 M LiCl, followed by a second
precipitation with 0.1 volume of sodium acetate, pH 5.2. Northern
blots
on Hybond N membranes were hybridized to random primer-labeled
probes
according to the manufacturer's instructions (Biolabs).
Blots were
stripped and reprobed with other probes to normalize
the amount of RNA
loaded.
Mapping of the PAS1 locus.
Restriction fragment
length polymorphism (RFLP) analysis was performed on 98 F8
recombinant inbred lines generated from a cross between the ecotypes
Landsberg erecta and Col0 (25) and digested with
HpaII. The BamHI genomic fragment flanking the
T-DNA right border was used as a probe in the Southern blot analysis. The linkage analysis was done with the MapMaker program. Recombination frequencies were calculated as described previously (25) and converted to map distances in centimorgans by using the Kosambi mapping
function (21).
Histochemical GUS assays.
Histochemical assays for
-glucuronidase (GUS) expression were performed as described by
Mollier et al. (32). The reactions were conducted for 4 to
8 h at 37°C for pas1-1 mutants and for 8 to 16 h
for pas1-1 heterozygous plants. Assays of GUS activity were
performed as described by Jefferson et al. (19), both on 9-day-old pas1-1 mutants and pas1-1/+ plants.
Functional complementation.
A 2.5-kb
XbaI-XhoI fragment corresponding to the
PAS1 full-length cDNA was cloned into the pKYLX71
(28) plant binary vector, previously digested with
XbaI-XhoI. Agrobacterium tumefaciens C58C1 (pMP90) was transformed by electroporation, and recombinant clones were isolated on kanamycin (20 µg/ml) and checked by Southern blot analysis. Plant transformations were performed by using the in
planta transformation system (2). Transformant plants were selected in vitro on kanamycin (100 µg/ml). The F2 and F3 progeny of
individual kanamycin-resistant plants were then analyzed for the
segregation of pas1 mutants.
Nucleotide sequence accession numbers.
The nucleotide
sequences of cDNA-A and cDNA-D have been deposited in GenBank under
accession no. U77365 and U77366, respectively.
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RESULTS |
Isolation of a pleiotropic mutant altered in early
development.
The pas1-1 mutant was isolated from the
T-DNA collection of INRA-Centre de Versailles as a heterozygous
pas1-1/+ line resistant to the Basta herbicide. A
pas1 allelic mutant (pas1-2) and several other
pas mutants representing three complementation groups were isolated from an ethyl methane sulfonate (EMS) mutant collection based
on their abnormal responses to exogenous CKs (12). Mutants grown in the dark had short and wide hypocotyls and lacked apical hooks
(Fig. 1A). For pas1 mutants
grown in the light, the phenotype was characterized by a very short and
thick hypocotyl and an altered cotyledon shape (Fig. 1B). The
pas1 mutants could not survive under normal growth
conditions but could be maintained in vitro. They often had fused
leaves with a vitreous appearance. Both pas1 allelic mutants
were characterized by the absence of secondary roots and a primary root
shorter than that of the wild type (Fig. 1B and C). After 3 months,
pas mutants developed abnormal compact and vitreous rosettes
(Fig. 1D). Several mutants produced finger-like structures before
growth was arrested. Some pas1 mutants were able to flower
but only developed very short stems with abnormal and sterile flowers
(data not shown).
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FIG. 1.
Phenotypes of pas1 mutants. (A) Seven-day-old
plants grown in the dark. Left, pas1-1 plant; right,
wild-type plant. (B) Plants 8 days after germination. From left to
right, wild type and three pas1-1 mutants. (C)
Three-week-old pas1-1 mutants. (D) Three-month-old
pas1-1 mutant. (E) Three-week-old wild type. (F) Wild-type
plants grown on 5 µM BA for 3 weeks. (G) pas1-1 mutants
grown in the absence (left) or in the presence (right) of 5 µM BA.
Plants were grown in the light unless otherwise indicated.
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While the growth of the wild type was severely inhibited by the
presence of 5 µM BA (Fig.
1E and F), the mutant displayed
a
hypertrophy of the apical part (Fig.
1G). We have previously
shown that
the response to CK of the
pasticcino mutants was
characterized
by an increase in cell divisions, specifically in the
apical part.
No significant difference between the
pas
mutants and the wild
type with regard to root growth inhibition in the
presence of
BA was observed (
12). The same results were
obtained with other
active CK molecules, such as zeatin and
1-(2-chloropyrid-4-yl)-3-phenylurea
(data not shown). The growth
responses of the
pas mutants in the
presence of other plant
hormones were analyzed in both light and
dark growth conditions, but
none of the hormones tested (auxin,
ethylene, gibberellic acid,
abscisic acid, and BR) was able to
induce a hypertrophy of the apical
parts of
pas mutants, indicating
that this particular
response was specific to CKs (
12).
Cytological analysis showed that
pas1-1 hypocotyls have
extra disorganized cell layers, irregular numbers of cortex cells,
a
loss of cell adhesion, and ectopic periclinal divisions in the
epidermis (Fig.
2A and B). The different
meristematic cell layers
were never clearly distinguishable. The
pas1-1 mutants had meristems
with a highly variable
structure, ranging from plants with almost
no meristem to those with a
very large meristem, which filled
the entire apical region (Fig.
2C, D,
and E).
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FIG. 2.
Effects of the pas1 mutation on cell
division. Hypocotyl cross sections of the wild type (A) and the
pas1 mutant (B) and longitudinal sections of the shoot
apical meristems of the wild type (C) and of pas1 mutants (D
and E). Bars, 200 µm (A, C, D, and E) and 400 µm (B). Sections were
made on 10-day-old seedlings.
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After three outcrosses of the
pas1-1 mutant with the wild
type, segregation was found to be consistent with
pas1-1
being a
nuclear, recessive, and monogenic mutation. In the progeny of
plants heterozygous for
pas1-1, the
pas1-1
mutation was shown
to cosegregate with a single T-DNA insertion
carrying the kanamycin
resistance gene (see Materials and Methods).
Isolation and molecular characterization of the PAS1
gene.
As genetic analysis of the segregation of the
Pas1-1
phenotype and the T-DNA insertion indicated tight
linkage between the T-DNA insert and the PAS1 gene, we
performed a molecular characterization of the pas1-1
mutation. Southern analysis of DNA extracted from pas1-1
mutants and probed with both the T-DNA right border (data not shown)
and left border (Fig.
3A)
revealed that the pas1-1 mutation was caused by the
insertion of a single T-DNA unit.
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FIG. 3.
Molecular characterization of the pas1-1
mutation. (A) Southern analysis of pas1-1 and wild-type
genomic DNA probed with the left T-DNA border (left) and with the
genomic fragment adjacent to the left T-DNA border (right). (B)
Schematic map of the T-DNA-tagged pas1-1 allele. The arrows
with the dashed lines show the two mRNAs transcribed from the
pas1-1 allele. The first transcript is transcribed from the
PAS1 promoter through the right T-DNA border giving rise to
the translational fusion between the 5' part of the PAS1
gene and the GUS gene. The second transcript arises due to
transcription from the cauliflower mosaic virus 35S promoter (35Sp)
through the BAR gene (which confers resistance to Basta) and
the 3' end of the PAS1 gene. The thin black line represents
the genomic DNA, and the T-DNA is shown by the black box. B,
BamHI; Bg, BglII; E, EcoRI; H,
HindIII; N, NsiI; P, PstI; S,
SacI; V, EcoRV; X, XbaI. a and b
denote the probes used for the Southern analysis. (C) GUS staining of
pas1-1/+ plants (a) and pas1-1 mutants (b), and
in a pas1-1/+ plant root (c) and in a pas1-1
plant root (d). The plants were grown for 9 days in standard conditions
in the light.
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We isolated different genomic DNA fragments adjacent to the right and
left borders of the T-DNA.
BamHI (4 kb) and
XbaI
(0.4
kb) genomic fragments were cloned as sequences flanking the right
border of the T-DNA by using the kanamycin plasmid rescue strategy
(
6) (see Materials and Methods). A 1.6-kb
XbaI
genomic fragment
adjacent to the T-DNA left border was isolated by an
inverse PCR
strategy (see Materials and Methods). Synthetic
oligonucleotides
corresponding to the genomic
XbaI
fragments, flanking both the
T-DNA right and left borders, were used in
a PCR on a wild-type
genomic-DNA template to verify whether the T-DNA
insertion had
caused any deletion or rearrangement (data not shown). A
sequence
analysis of the T-DNA insertion site in the
pas1-1
mutant revealed
a deletion of 14 bp in the
PAS1 gene.
In the pGKB5 binary vector used to generate the T-DNA collection
(
5), the ATG of the promoterless
uidA gene is 40 bp downstream
of the T-DNA right border, with no in-frame stop codon.
An analysis
of the genomic sequence flanking the T-DNA insert revealed
an
open reading frame (ORF) adjacent to the right border insertion
sequence, in frame with the methionine initiation codon of the
promoterless
uidA (
GUS) gene (Fig.
3B). This
indicated that a
translational fusion of the
pas1-1-tagged
allele with the
uidA coding sequence had occurred, an event
which is consistent with
the GUS staining of the
pas1-1
mutant (Fig.
3C). In further agreement
with this, Northern blot
analysis of
pas1-1 RNA identified a 3.4-kb
transcript that
hybridized with both a
uidA and a
PAS1 probe
(data
not shown).
The two
XbaI fragments adjacent to the right and left T-DNA
borders were used as probes to screen both a wild-type
Arabidopsis genomic library (EEC-BRIDGE
Arabidopsis DNA Stock Center) and
a cDNA
Arabidopsis library (
31). The genomic sequence of
the
PAS1 gene was obtained by sequencing both the
BamHI and the
XbaI
clones. Synthetic
oligonucleotides corresponding to the genomic
sequence allowed us to
amplify and sequence the same region from
a wild-type (WS) template.
The analysis of the genomic sequence
and its comparison with the cDNA
sequence revealed that the
PAS1 gene was interrupted by 18 introns and that the entire gene was
4.2 kb long (Fig.
5A). The T-DNA
was inserted before the last
intron, 1,747 nucleotides from 5' end of
the cDNA. All the introns
identified in the
PAS1 gene showed
the plant canonical acceptor
and donor splice sites (NetPlantGene mail
server:
www.cbs.dtu.dk/NetPlantGene.html).
Several independent cDNA
clones were isolated. Among them, cDNA-D
was chosen as containing the
full-length
PAS1 transcript, because
Northern blot analysis
of RNA from 9-day-old wild-type seedlings
showed a transcript of the
expected size (data not shown). A sequence
analysis of cDNA-D revealed
an ORF of 1,902 bp, corresponding
to a protein of 634 amino acids (69.7 kDa). The first ATG in the
ORF was preceded by several in-frame stop
codons, suggesting that
this is indeed the start codon. The full-length
cDNA and the partial
cDNA clones had poly(A) tails, although they
differed slightly
in the length of the 3' untranslated region, perhaps
due to the
presence of different polyadenylation sites (Fig.
4 and
5A).
Another
class of
PAS1 cDNA (cDNA-A), which contained an
insert 70 bp longer
than that of cDNA-D, was identified. A sequence
comparison of
the two cDNA classes (A and D) revealed that this
difference was
due to the lack of splicing of the second intron in the
cDNA-A
class (Fig.
4). As the possibility exists that the cDNA-A
represents
an aberrant
PAS1 unspliced transcript, we focused
our attention
on cDNA-D. However, it is possible that the cDNA-A class
results
from a differential splicing, as more than one independent
clone
of the cDNA-A type was found in the library. Human FKBP12 is
encoded
by different mRNAs, varying in abundance and 3' untranslated
region,
deriving from the differential splicing of five exons (
1,
33).
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FIG. 4.
PAS1 cDNA sequence and predicted amino acid
sequence. The sequences of both cDNA-A and cDNA-D are shown. The 5'
ends, the start codons, and the 3' ends of the two classes of cDNAs are
indicated by bold face, italic, uppercase letters (cDNA-D) and by
underlined letters (cDNA-A). The unspliced intron in cDNA-A (70 nucleotides) is indicated by lowercase letters. The two putative
cdc2-type protein kinase C phosphorylation sites are boxed, while the
Tyr phosphorylation site is indicated by a boldface broken line. The
putative NLSs are underlined. The point mutations in pas1-2
at nucleotides 417 and 1317 are indicated in boldface, and altered
residues are circled.
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FIG. 5.
Structure of the PAS1 gene and of the PAS1
protein. (A) Schematic representation of the PAS1 gene.
PAS1 contains 19 exons, represented by the black boxes, and
18 introns, shown as hatched boxes. The vertical arrow shows the
location of the first ATG codon of cDNA-D. The arrowhead indicates the
T-DNA insertion site. The 5' end of the cDNA is shown by the horizontal
arrow. Abbreviations for restriction sites are as defined in the legend
for Fig. 3. (B) PAS1 predicted protein. The black box indicates the
putative bipartite NLS in the N-terminal region. The two hatched boxes
show FKBP-like domains I and II. The three shaded boxes indicate the
TPR domains. The positions of the amino acid substitution (K N) and
the stop codon (WSTOP) for the pas1-2 allelic mutant are
also indicated.
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The
PAS1 cDNA of the
pas1-2 allelic mutant was
sequenced. A comparison with the wild-type gene sequence revealed a
G-to-A
point mutation at nucleotide 1317 (starting from the 5' end),
which creates a translational stop codon (W397STOP), and a G-to-C
point
mutation at nucleotide 417, which causes a lysine-to-asparagine
substitution (K87N) within the first FKBP domain (Fig.
4 and
5B).
The
PAS1 gene has been mapped by RFLP analysis with the
genomic-DNA fragment adjacent to the T-DNA right border as a probe
(Fig.
3B). The
PAS1 gene is located on chromosome 3 at 106.6 centimorgans
(marker ve042) on the recombinant inbred map
(
25).
Complementation of the pas1-2 mutant.
We wished to
determine whether the mutation in the PAS1 gene was
responsible for the Pas1-1 phenotype. The coding region
of the full-length PAS1 cDNA-D was cloned into plant binary
vector pKYLX71 (28) under the control of the cauliflower
mosaic virus promoter (35S2). A. tumefaciens C58C1 (pMP90) transformants were selected on kanamycin and used to
transform plants, which were heterozygous for the pas1-2
mutation, by the in planta Agrobacterium-mediated
transformation method (2). Kanamycin-resistant T1 plants
were allowed to self-pollinate. If the cDNA was able to complement the
pas1-2 mutation in the progeny of the heterozygous
transformed plant, we expected to obtain only 1 kanamycin-sensitive
mutant plant for every 15 wild-type plants (12 kanamycin-resistant and
3 kanamycin-sensitive plants). The segregation analysis was performed
in vitro on a kanamycin-supplemented medium and revealed that several
independent transformants, heterozygous for the pas1
mutation, segregated with the expected ratio. The segregation analysis
of four such transformants is shown in Table 1. Some of the transformants segregating
as wild-type kanamycin-resistant plants showed abnormal developmental
phenotypes. Whether this is due to the overexpression of
PAS1 or to a phenomenon of cosuppression of the
PAS1 gene in a wild-type background has yet to be
determined. However, we never found any pas1 mutant plant
resistant to kanamycin in the progeny of the transgenic plants
analyzed.
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TABLE 1.
Functional complementation of the pas1-2
mutant as indicated by a segregation analysis for the
pas1 mutanta
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PAS1 has similarities to the immunophilin proteins.
The
predicted amino acid sequence of PAS1-D was compared with sequences in
current databases (National Center for Biotechnology Information; BLAST
network server). The PAS1 protein has significant similarity to FKBPs
belonging to the family of immunophilins (reviewed in reference
20). The term FKBP refers to any protein which binds
both FK506 and rapamycin immunosuppressive compounds. Several FKBPs
have been isolated to date from a broad range of organisms, and they
are named according to their molecular masses (13, 42).
These proteins also have peptidyl-prolyl cis-trans isomerase (rotamase) activity.
The highest scores for the PAS1 protein were obtained with two plant
protein sequences: that of the ROF1 protein from
Arabidopsis (FKBP62) (
41) and that of wFKBP73 from
Triticum
aestivum (
3),
two high-molecular-weight FKBPs. PAS1 has
71% similarity and 31%
identity with
Arabidopsis ROF1 and
54% similarity and 31.5% identity
with wheat wFKBP73. The PAS1
protein also has sequence similarity
with several FKBP52 (p59) proteins
from mammals (
23,
34,
40).
The overall degree of sequence
similarity of PAS1 with FKBP52
from
Homo sapiens,
Mus
musculus, and
Oryctolagus cuniculus (accession
no.,
Q02790,
X70887, and
M84474, respectively) ranges
from 51 to 49% (28 to
24% sequence identity).
Human FKBP52 contains three FKBP-like domains, but only the N-terminal
domain has FKBP-type rotamase activity and the FK506
binding site. Of
the 14 FK506-binding residues found in human
FKBP12, 13 are conserved
in the first FKBP domain of human FKBP52
(
20). An alignment
of PAS1 with human FKBP52 revealed the presence
of an N-terminal
FKBP-like domain which showed conservation of
4 of the 14 residues
involved in FK506 binding (R77, I91, Y117,
and F130) (Fig.
5A). Six
residues are identical to those resulting
from substitutions at
corresponding positions in other FKBPs (i.e.,
V67 in
E. coli
FKBP22, E68 in
E. coli FKBP16, I81 in
Saccharomyces cerevisiae FKBP13, K89 in human FKBP25, L94 in
E. coli
FKBP22,
and A126 in human FKBP13) (
20). Some substitutions
within the
FK506 binding site are conservative substitutions (e.g.,
E68D
and M90V). The C-terminal region of PAS1 is characterized by a
tetratricopeptide repeat (TPR) domain: a 34-amino-acid repeat
present
in multiple arrays. There are three such arrays in PAS1
and in FKBP52
(Fig.
5B and
6B). These domains have been
proposed
to form amphipathic
-helices that mediate protein-protein
interactions
(reviewed in references
16 and
22).
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|
FIG. 6.
Alignments of the FK506 binding domain and of the TPR
region of PAS1. (A) Comparison of the FK506 binding domain (domain I)
of PAS1-D with that of hFKBP52. The first FKBP domain in hFKBP52 spans
amino acids 32 to 136. The underlined amino acids are the residues
required for FK506 binding in hFKBP12. The corresponding residues in
PAS1 are indicated by vertical lines if they are identical, by boldface
type if they are identical to corresponding residues in other FKBPs, or
by lowercase letters if they are different. The horizontal lines
indicate gaps introduced to maximize alignment. (B) Amino acid sequence
similarity in the three units of the TPR domain. The sequences compared
to PAS1 are ROF1 from A. thaliana (FKBP62), wFKBP73
(TAFKBP70) from T. aestivum, and hFKBP52. Light gray shading
indicates similar residues, while identical amino acids are printed in
white. The three TPR units are underlined. The alignments were
generated with the PILEUP program of the Genetics Computer Group
package.
|
|
An analysis of the predicted amino acid sequences by the PROSITE method
revealed the presence of different putative phosphorylation
sites in
the PAS1 protein. Among these are two cyclic AMP/cyclic
GMP
phosphorylation sites, one site for casein kinase, and several
protein
kinase C phosphorylation sites, two of which correspond
to the cdc2
type. One putative tyrosine phosphorylation site is
also present in
PAS1 (Fig.
4). In the N-terminal region of PAS1-D,
a putative bipartite
nuclear localization signal (NLS) is present
within an uncharged,
hydrophobic region (Fig.
4 and
5B).
The PAS1 gene is expressed throughout the plant and is
regulated by CK.
In order to study the mechanism by which the PAS1
protein contributes to development, we analyzed PAS1 gene
expression. Total RNA was extracted from different organs of wild-type
and pas1-1 heterozygous plants and hybridized with a
PAS1 cDNA probe. This revealed that PAS1 was
expressed in stems, leaves, flowers, siliques, and roots in wild-type
plants (Fig. 7A) and in pas1-1
heterozygous plants (data not shown). As a result of the translational
fusion with the uidA gene, the pas1-1 transcript
size was approximately 3.4 kb (Fig. 3B). The PAS1-GUS
translational fusion allowed us to perform a study of PAS1-1
expression by histological analysis, on both pas1-1
homozygous and heterozygous plants. The pas1-1 mutant showed
strong GUS staining in all of the apical part, in the vascular cylinder
of the root, and in the root tip (Fig. 3C, panels b and d). This
expression was not affected by light or dark growth conditions or by
the seedling developmental stage. GUS staining in the
pas1-1/+ plants showed a strong staining of the apical and
root meristematic regions and no detectable staining in the cotyledons,
leaves, hypocotyls, or roots under all conditions tested (Fig. 3C,
panels a and c).
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|
FIG. 7.
PAS1 expression analysis. (A) RNAs were
isolated from different organs of soil-grown wild-type plants, except
for roots, which were prepared from plants grown in vitro. F, flowers;
cL, cauline leaves; rL, rosette leaves; S, siliques; St, stem; R,
roots. (B) PAS1 induction by BA in the wild type. RNAs were
extracted from 9-day-old Col0 BA-treated plants (0, 103,
102, 101, 1, and 10 µM BA). As a control
for the loading of RNA samples, the blot was also hybridized with an
actin cDNA probe. (C) PAS1 expression in the
pas1-1 mutants. RNAs were extracted from 9-day-old mutants
and wild-type WS grown on increasing amounts of BA (0, 0.1 and 5 µM).
Arrowheads: a, PAS1-GUS chimeric mRNA; b, wild-type
PAS1 mRNA; c, chimeric mRNA transcribed from the 35S
promoter through the bar gene and the 3' end of the
PAS1 gene (Fig. 3B). (D) PAS1 expression in the
pas1-2 allele and in the other two pasticcino
mutants, pas2 and pas3-1. Because these mutants
are in the Col0 ecotype background, wild-type Col0 was used as the
control. RNAs were extracted from 9-day-old BA-treated plants (0, 0.1, and 5 µM BA). In panels C and D the 25S rDNA was used to control the
loading of RNA samples. Fifteen micrograms of total RNA was loaded in
each lane.
|
|
We also investigated CK regulation of
PAS1 gene expression
in 9-day-old wild-type plants. The steady-state level of
PAS1 transcripts
was higher in wild-type (ecotype Columbia)
plants grown on BA
concentrations up to 5 µM than in untreated
controls (Fig.
7B).
We have previously shown that the
pas
mutants display a normal
response to auxin (
12). We analyzed
total RNA extracted from
9-day-old wild-type plants grown on 5 µM
picloram, an auxin analog.
The results revealed that the steady-state
level of
PAS1 mRNA
was weakly increased by auxin, but to a
much lesser extent than
by CK (data not shown).
The pas mutants show altered expression of the
PAS1 gene.
PAS1 gene expression in the two
allelic pas1 mutants and in the pas2 and
pas3 mutants (12), either in the presence or
absence of exogenous CKs (BA) was analyzed.
In
pas1-1 mutants two transcripts can be detected due to the
insertion of the T-DNA (see above), but the transcript of the
wild-type
size is absent (Fig.
7C). In heterozygous
pas1-1 plants
the
wild-type transcript is present with the two other transcripts
(data
not shown).
PAS1 mRNA levels were still increased by CK
in
the
pas1-1 mutant, but the response was shifted towards
lower
BA concentrations compared to the response of the corresponding
wild type (ecotype WS) (Fig.
7C). This result was confirmed by
a
fluorimetric GUS quantitative analysis.
PAS1 expression was
still induced by BA, both in
pas1-1 homozygous and
heterozygous
plants, but the peak of BA induction of GUS activity was
reached
at 0.1 µM BA (data not shown). In
pas1-2 plants, a
transcript
of the wild-type size is present and is constitutively
expressed
(Fig.
7D).
PAS1 expression was affected in
pas2-1 and
pas3-1 mutants. For
pas2-1 mutants, the
PAS1 mRNA could be detected in untreated
mutants and in
mutants grown in the presence of 0.1 µM BA but
not in presence of 5 µM BA (Fig.
7D). For
pas3-1 mutants,
PAS1 mRNA
could not be detected unless the mutants were grown at a
low BA
concentration (0.1 µM) (Fig.
7D). These results suggest
that the CK
sensitivity of
PAS1 expression is modulated by the
other two
PAS genes, which may be required for its controlled
expression.
| |
DISCUSSION |
PAS1 is involved in the control of cell
proliferation.
We have previously reported the isolation of three
classes of mutants with very similar pleiotropic phenotypes
characterized by the presence of large, abnormal meristems leading to
disorganized rosettes made of fused, vitreous leaves (12).
The pas mutants were shown to be particularly altered in
their response to CKs (12). The three PAS genes
are also involved in the control of embryogenesis. Both
pas1-1 and pas1-2 mutants contain mutations (T-DNA insertion and point mutations, respectively) in the coding sequence of the PAS1 gene. The fact that pas1-2
can be complemented with the wild-type PAS1 cDNA confirms
that the gene corresponding to the mutant phenotype has indeed been
cloned. The expression of the PAS1 gene, which is impaired
in pas1 mutants, is also affected in pas2 and
pas3 mutants, suggesting a possible molecular basis for the
similarities of their phenotypes. These results tend to confirm the
previous biochemical analysis of the pas mutants, which
demonstrated that pas mutants were biochemically closely linked (12).
The
pas mutants are an example of plant mutants which have a
general deregulation of the control of cell proliferation. We
hypothesize that this deregulation is due to the absence of a
functional PAS1 protein, which in the wild type would antagonize
cell
proliferation. It is proposed that the PAS1 protein accumulates
and
functions in dividing tissues, such as the meristems, to prevent
uncontrolled cell division. In
pas1-1/+ plants, the PAS1-GUS
protein
accumulates preferentially in the meristematic area. In the
wild
type, posttranscriptional regulation possibly occurs to prevent
the production of the protein in all the organs (the
PAS1
mRNA
is not organ specific). However, in
pas1-1 plants, the
chimeric
PAS1-GUS protein is overproduced not only in the meristematic
zones but also in all the tissues undergoing cell division. Possibly
the lack of functional PAS1 in the
pas1 mutant causes
ectopic
cell proliferation, which in turn induces ectopic
PAS1 expression
in a regulatory feedback loop.
The cellular proliferation in
pas1-1 plants is enhanced
specifically by CKs. In wild-type plants
PAS1 expression is
up-regulated
by CKs, and in
pas1-1,
pas1-2, and
pas2-1 plants, CK regulation
of
PAS1 expression
is altered. Although we previously showed that
only CKs have an effect
on the
pas phenotype (
12), the possibility
exists
that the action of CKs is mediated by other hormones, such
as auxins
and BR, which are known to interact with CKs in plant
development.
Therefore, PAS1 may function directly in a CK pathway
controlling cell
division or in a pathway controlling similar
downstream events.
PAS1 is an FKBP-like protein with TPR domains.
The PAS1
protein has significant sequence similarity with the immunophilin
family of FKBPs. Two immunophilin families can be distinguished:
cyclophilins and FKBPs. The FKBPs, represented by the well-studied
FKBP12, bind the immunosuppressants FK506 and rapamycin, whereas the
cyclophilins bind cyclosporin A (42). Although the two
families do not have structural or sequence homology, all immunophilins
identified to date exhibit peptidyl-prolyl cis-trans isomerase (rotamase) activity. Immunophilins are housekeeping proteins,
highly conserved throughout evolution, which may mediate critical
cellular functions (reviewed in reference 20).
Several reports have shown the presence of immunophilins in plants
(3, 7, 14, 18, 24). FKBPs with distinct subcellular
localizations are known (7, 24, 27; reviewed in
reference 20). PAS1 has a nuclear-localization
signal, and it is likely, although as yet untested, that PAS1 functions
in the nucleus.
Recently, two low-molecular-weight FKBPs were identified in
A. thaliana (AtFKBP15-1 and AtFKBP15-2) (
26) and two
high-molecular-weight
FKBPs were identified in both
A. thaliana (ROF1 or FKBP62) (
41)
and wheat (wFKBP73)
(
3). The PAS1 protein has the most similarity
to the large
FKBPs such as
Arabidopsis ROF1, wheat wFKBP73, and
the
mammalian FKBP52 and FKBP51. All these proteins have N-terminal
FKBP
domains and C-terminal TPRs.
Only the most N-terminal of the FKBP domains of human FKBP52 has
rotamase activity and a binding site for immunosuppressants
FK506 and
rapamycin (
17,
39). Wheat FKBP73 has rotamase activity,
which can be inhibited by FK506 and rapamycin (
3). Such
binding
properties are currently being tested with the PAS1 purified
protein.
Preliminary tests with FK506 on wild-type
Arabidopsis seedlings
showed that the drug only affected
seedling development at high
concentrations and did not induce the
Pas1
phenotype. This may be due to a lack of penetration
of FK506
into the plants. Further experiments on protoplasts or cell
cultures
will be performed in order to study the effects of these
molecules
in plants and to identify their cellular targets.
FKBP52 was identified as a component of unliganded steroid hormone
receptor complexes, along with the heat shock proteins
hsp90 and hsp70
(
4,
8,
34,
40,
43). The TPR domain
is necessary for FKBP52
to interact with hsp90 in the steroid
receptor complex
(
35; reviewed in reference
37).
The TPR motif
is a 34-amino-acid motif of variable sequence which has
defined
a protein family involved in cell cycle regulation, RNA
synthesis,
protein transport, Ser-Thr dephosphorylation, and the heat
shock
response (
16,
22). Interestingly, the TPR domains of
mammalian
FKBP52 have a high degree of similarity, in position and
amino
acid composition, to the PAS1 TPR domains. We found two mutations
in the
pas1-2 cDNA sequence. The G-to-A transversion is
characteristic
of EMS-induced mutations and causes a truncation of the
PAS1 protein
prior to the TPR domain, similar in position to the
alteration
caused by the insertion of the T-DNA into
pas1-1.
The G-to-C mutation
causes a single-amino-acid substitution within the
FKBP domain.
It is likely then that the TPR domain, which is absent
from the
predicted proteins encoded by both
pas1-1 and
pas1-2 alleles,
is of particular importance in the function
of PAS1. Whether the
PAS1 protein is able to bind hsp90 and the
identities of other
potential binding partners have yet to be
determined.
As the PAS1 gene encodes an FKBP-like protein similar to steroid
receptor-associated FKBPs, it should be noted that the BR,
which are
plant growth factors, exhibit structural similarity
to animal steroid
hormones (
11). Bioassays on both intact plants
and hypocotyl
segments have shown that a broad spectrum of cellular
responses is
elicited by BR. Although the action of BR in the
elongation of stem
tissues has been well characterized, their
effects on cell division and
cell differentiation have also been
reported. BR are known to act
synergistically with auxin in stimulating
cell elongation
(
38), and there is evidence that BR action affects
the
content of endogenous auxin. BR-induced changes in auxin level
may
alter the auxin/CK ratio in plant tissues (
15).
pas1 mutants represent the first inactivation of a gene
encoding an FKBP-like protein in higher eukaryotes. Genetic studies
on
immunophilin gene disruption in microorganisms have been performed
(reviewed in reference
10). Despite the widespread
occurrence
and strong sequence conservation of immunophilin genes,
their
inactivation has been shown to have little or no effect on cell
viability. This work shows that when the expression of an FKBP-like
gene is impaired, as in
pas mutants, the control of cell
proliferation
is affected.
In conclusion, we believe that the
pas1 mutants represent a
useful tool to understand the role of immunophilin-like proteins
in
plant development and to unravel their potential functions
in plant
hormonal signalling pathways.
| |
ACKNOWLEDGMENTS |
P.V. was supported by an INRA postdoctoral fellowship from DSPV.
We are grateful to G. Pelletier and his group for providing the T-DNA
lines, to B. Courtial for RFLP analysis, and to T. Desprez for
technical assistance with software analysis. The A. thaliana genomic and cDNA libraries were kindly provided by J. Mulligan and C. Robaglia, respectively. We also thank E. E. Baulieu and his group
for helpful discussions and for the gift of the FK506 and rapamycin
molecules and Heather Mckhann and Isabelle Barlier for critical reading
of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie Cellulaire, INRA-Centre de Versailles, Route de St. Cyr, 78026 Versailles Cedex, France. Phone: (33 1) 30833095. Fax: (33 1) 30833099. E-mail: bellini{at}versailles.inra.fr.
Present address: Dipartimento di Genetica e Biologia Molecolare,
Università di Roma "La Sapienza," 00185 Rome, Italy.
| |
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Abstract
Introduction
