The Plant Cell, Vol. 15, 1578–1590, July 2003, www.plantcell.org © 2003 American Society of Plant Biologists
The Arabidopsis Cupin Domain Protein AtPirin1 Interacts with
the G Protein -Subunit GPA1 and Regulates Seed Germination
and Early Seedling Development
Yevgeniya R. Lapik and Lon S. Kaufman1
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607
Heterotrimeric G proteins are implicated in diverse signaling processes in plants, but the molecular mechanisms of their
function are largely unknown. Finding G protein effectors and regulatory proteins can help in understanding the roles of
these signal transduction proteins in plants. A yeast two-hybrid screen was performed to search for proteins that interact
with Arabidopsis G protein -subunit (GPA1). One of the identified GPA1-interacting proteins is the cupin-domain protein
AtPirin1. Pirin is a recently defined protein found because of its ability to interact with a CCAAT box binding transcription
factor. The GPA1–AtPirin1 interaction was confirmed in an in vitro binding assay. We characterized two atpirin1 T-DNA insertional mutants and established that they display a set of phenotypes similar to those of gpa1 mutants, including reduced
germination levels in the absence of stratification and an abscisic acid–imposed delay in germination and early seedling development. These data indicate that AtPirin1 likely functions immediately downstream of GPA1 in regulating seed germination and early seedling development.
INTRODUCTION
Heterotrimeric G protein–mediated signal transduction is one of
the most conserved eukaryotic signaling mechanisms and is
responsible for the transmission of extracellular signals to intracellular effectors. Heterotrimeric guanine nucleotide binding
proteins (G proteins) belong to the superfamily of regulatory
GTP hydrolases (Kaziro et al., 1991). G proteins consist of three
subunits: (G), (G), and (G). In its inactive state, the Gsubunit is bound to a GDP molecule and is associated with a
G-subunit. Upon activation by a G protein–coupled receptor,
the G-subunit exchanges GDP for GTP and dissociates from
the G-subunit. As a result, the G- and G-subunits are
free to transduce the signal to intracellular effectors (reviewed
by Sprang, 1997; Hamm, 1998). In animal systems, effector
molecules include adenylyl and guanylyl cyclases, phosphodiesterases, phospholipases, and phosphoinositide 3-kinases
(reviewed by Spiegel et al., 1994; Marinissen and Gutkind,
2001). Little is known about plant G protein effectors. Recently,
a small GTPase Rac1 was implicated in the downstream functioning of the rice G-subunit during the plant’s pathogen response (Suharsono et al., 2002).
In contrast to animals, in which multiple -, -, and -subunits have been found, plant G protein subunits are encoded
by single- or double-copy genes. Two G -subunit–encoding
genes were found in pea (Marsh and Kaufman, 1999), tobacco
(Saalbach et al., 1999; Ando et al., 2000), and soybean (Kim et
al., 1995; Gotor et al., 1996). The Arabidopsis genome appears
to contain only one gene that encodes a canonical G -subunit
1 To
whom correspondence should be addressed. E-mail lkaufman@
uic.edu; fax 312-413-2691.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.011890.
(Ma et al., 1990), one gene that encodes a G -subunit (Weiss et
al., 1994), and two genes that encode G -subunits (Mason
and Botella, 2000, 2001). Nevertheless, a number of largely
pharmacological studies have suggested plant G protein involvement in a variety of processes, including blue light–mediated responses (Warpeha et al., 1991), red light–mediated responses (Romero and Lam, 1993), abscisic acid (ABA) and
gibberellin signaling, regulation of ion channels, and pathogen
resistance (reviewed by Assmann, 2002).
Recently, two independent T-DNA insertions that are likely to
be null mutations were found in the Arabidopsis G -subunit–
encoding gene GPA1 (Ullah et al., 2001). Analysis of these gpa1
mutants revealed that they have reduced cell division during
hypocotyl and leaf formation (Ullah et al., 2001). Additionally,
gpa1 mutants are impaired in the inhibition of stomatal opening
by ABA (Wang et al., 2001). The gpa1 mutant seeds appear to
be hypersensitive to sugars, hyposensitive to gibberellic acid,
and insensitive to brassinosteroids. Furthermore, gpa1 mutant
seeds exhibit less than wild-type germination levels in the presence of exogenous ABA (Ullah et al., 2002). Multiple phenotypes of gpa1 mutants suggest that GPA1 is an integration
point of several signaling pathways. It is likely that GPA1 executes its diverse functions by interacting with a variety of regulatory and effector proteins; however, no GPA1-interacting proteins have been reported to date.
To further investigate the mechanism of G -subunit function
in Arabidopsis, we performed a yeast two-hybrid screen to
identify its interacting partners. One of the GPA1-interacting
proteins found in the screen was AtPirin1, an ortholog of the
human pirin protein. Pirin is a recently defined protein found as
a result of its ability to interact with the CCAAT box binding
transcription factor NFI/CTF1 (Wendler et al., 1997). We have
established that AtPirin1 interacts specifically with GPA1 in
AtPirin1/GPA1 Role in Seed Germination
1579
yeast as well as in vitro. AtPirin1 transcript levels were found to
be upregulated by ABA and low-fluence red light but not by
low-fluence blue light. Two Arabidopsis mutants harboring
T-DNA insertions in the AtPirin1 gene were characterized. It
was observed that insertions in the AtPirin1 gene cause reduced levels of seed germination in the absence of stratification and that atpirin1 mutant seed germination is hypersensitive
to exogenous ABA in a manner similar to that of gpa1 mutants.
Additionally, we observed that atpirin1 mutant plants initiate
primary shoots and flower earlier than wild-type plants.
RESULTS
The Arabidopsis G-Subunit Interacts with AtPirin1
in the Yeast Two-Hybrid Screen
To identify potential components of G protein–mediated signaling pathways in plants, we performed a yeast two-hybrid
screen of the Arabidopsis seedling library using a full-length Arabidopsis G-subunit (GPA1) as “bait.” Screening of 1.9 106 transformants yielded 16 positive clones that specifically
activate His3 and LacZ reporter genes. Restriction pattern
comparison indicated that four of these positive clones contained partial cDNAs of the same gene. Sequence analysis revealed a protein homologous with human pirin (Wendler et al.,
1997). Therefore, we named this GPA1-interacting protein
AtPirin1. The remaining GPA1-interacting clones will be described elsewhere. AtPirin1 clones caused specific activation
of His3 and LacZ reporter genes and did not interact with the
lamin protein used as a control bait (Figure 1A). The interaction
between the Arabidopsis proteins ETR1 and CTR1 (Clark et al.,
1998) served as a positive control for the assay (Figure 1A). The
two shortest GPA1-interacting AtPirin1 cDNA clones corresponded to the 170 C-terminal amino acids of the AtPirin1 protein, indicating that the first 117 N-terminal amino acid residues
are not critical for the interaction with GPA1 (Figure 2A). The
two longest interacting AtPirin1 cDNA clones lack the 64 N-terminal amino acid residues (Figure 2A).
AtPirin1 Interacts with GPA1 in Vitro
To confirm that AtPirin1 binds directly to GPA1, we studied
their interactions using an in vitro binding assay. For these experiments, the GPA1 coding region was overexpressed as a
glutathione S-transferase (GST) fusion in Escherichia coli and
immobilized on glutathione-Sepharose beads. Beads coated
with GST alone served as a control (Figure 1B). In vitro binding
assays were performed with AtPirin1 obtained by coupled in
vitro transcription/translation. After multiple washes, bound
proteins were separated on SDS-containing polyacrylamide
gels. Autoradiographic detection revealed that AtPirin1 interacts with GPA1 and not with GST alone (Figure 1C).
Additionally, we tested whether AtPirin1 interaction with GPA1
depends on the GPA1 activation state. For this analysis, we examined the in vitro activation of GPA1 by incubation with GTP S
(a nonhydrolyzable GTP analog) before the binding assay. In this
experiment, AtPirin1 displayed no significant preference for either the active or the inactive form of GPA1 (data not shown).
Figure 1. Interaction between AtPirin1 and GPA1.
(A) Reconstitution of the yeast two-hybrid interaction between AtPirin1
and GPA1. The indicated combinations of the bait (pLexA-NLS) and
prey (pACT) constructs were transformed into the yeast reporter strain.
Transformants were examined for -galactosidase activity in the presence of 5-bromo-4-chloro--D-galactoside (X-gal) and for growth in
the absence of His (His).
(B) Coomassie blue staining of the GST-GPA1 fusion protein and GST
(positions of the GST-GPA1 and GST proteins are indicated by arrows).
MW, molecular mass.
(C) In vitro protein interaction between AtPirin1 and GPA1. GST-GPA1
was overexpressed in E. coli, immobilized on glutathione-Sepharose 4B
beads, and incubated with 35S-labeled AtPirin1 protein obtained by coupled in vitro transcription/translation. AtPirin1 binds to GST-GPA1 but
not to GST alone.
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AtPirin1 Is a Conserved Protein Belonging
to the Cupin Superfamily
Conceptual translation of AtPirin1 cDNA revealed that it encodes a 287–amino acid protein. Database searches identified
several more pirin genes in the Arabidopsis genome. AtPirin1
is closely related to two of its family members, AtPirin2 and
AtPirin3, with 66% identity and 75% amino acid similarity (Figure 2A). Sequence alignments revealed that pirin is present
in mammals, plants, and a variety of prokaryotic species,
whereas there are no putative pirin orthologs in three model
Figure 2. AtPirin1 Amino Acid Sequence Analysis, AtPirin1 Gene Structure, and Locations of the T-DNA Insertions.
(A) Alignment of the deduced amino acid sequences of AtPirin1, AtPirin2, AtPirin3, AtPirin4, tomato pirin (TOMpirin), and human pirin (HUMpirin). The
alignment was generated using the CLUSTAL X program with default parameters. Identical amino acids are highlighted with a black background, and
similar residues are highlighted with a gray background. Regions corresponding to motifs 1 and 2 of the cupin domain are indicated. The single and
double asterisks mark residues corresponding to the beginning of the longest and shortest GPA1-interacting AtPirin1 clones, respectively, identified
in the two-hybrid screen. Arrows indicate the positions of T-DNA insertions in the atpirin1-1 and atpirin1-2 mutants.
(B) Scheme of the AtPirin1 gene. Relative positions of potential cis elements mentioned in the text are shown. The positions of the atpirin1-1 and
atpirin1-2 T-DNA insertions are indicated. Boxes represent exons, and motifs 1 and 2 of the cupin domain are marked by vertical and horizontal
hatching, respectively.
AtPirin1/GPA1 Role in Seed Germination
1581
eukaryotic species with fully sequenced genomes: Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila
melanogaster.
Pirin does not appear to have any known functional domains;
however, it contains a two-motif sequence characteristic of the
cupin domain (Figure 2A). Based on the three-dimensional
structure of several cupin proteins, it was established that the
two-motif cupin domain contains residues implicated in forming a -barrel fold and in metal binding (Gane et al., 1998;
Dunwell et al., 2000). The -barrel fold also is associated with
the high levels of thermal stability of cupin superfamily members (Dunwell et al., 2001). The superfamily of cupin proteins is
the most functionally diverse of any family described to date
and includes enzymes, transcription factors, seed storage proteins, auxin binding proteins, stress-related proteins (spherulins
and germin-like proteins), and other proteins (Dunwell et al.,
2000, 2001).
The fact that the two shortest GPA1-interacting Atpirin1
cDNA clones found in the two-hybrid screen do not contain the
cupin domain (Figure 2A) indicates that the cupin domain is not
required for interaction with the G -subunit.
AtPirin1 mRNA Levels Are Regulated by Abscisic Acid
and Red Light
Sequence analysis of the 5 regulatory region of AtPirin1 revealed the presence of several potential cis-acting regulatory
elements, including two ACGT elements and one TT motif at
308, 430, and 512 bp upstream of the AtPirin1 ATG codon
(Figure 2B). ACGT elements usually are defined as 8- to 10-bp
sequences with the core sequence ACGT (Busk and Pages,
1998; Leung and Giraudat, 1998); they include the ABRE-like
elements (abscisic acid–responsive elements) and the G-box
elements. ABREs are found in numerous promoters of ABAand stress-inducible genes (Leung and Giraudat, 1998). The
strong ABRE (CACGTG), found 430 bp upstream of the
AtPirin1 gene ATG codon, is identical to the core sequence of
the G-box element. G boxes are found in promoters of several
light-regulated genes, such as CAB, RBCS, and CHS (Busk
and Pages, 1998).
In contrast to the ACGT elements, little is known about the
TT motif. The TT motif, also called the TCGT motif (TTTCGTGT),
was found originally in the distal promoter region of the desiccation- and ABA-inducible Dc3 gene of carrot and appears to
be involved in ABA-induced gene expression in vegetative tissues (Busk and Pages, 1998).
The finding of potential cis-regulatory elements in the promoter of the AtPirin1 gene prompted us to test the effects of
ABA and different light conditions on AtPirin1 transcript levels.
RNA gel blot analysis of poly(A) RNA did not reveal any transcript; therefore, we performed relative quantitative reverse
transcription (RT)–PCR.
RT-PCR revealed that AtPirin1 is present at similar levels in
Arabidopsis seedlings and mature plants (data not shown). The
effect of ABA on AtPirin1 transcript levels was tested by spraying 9-day-old plants with 100 M ABA and harvesting tissue 4 h
later. This ABA treatment increased AtPirin1 transcript levels
(Figure 3A). The increase was approximately fivefold, as deter-
Figure 3. AtPirin1 Transcript Levels Are Regulated by ABA and LowFluence Red Light.
(A) Transcript levels of the AtPirin1 gene are induced by ABA. AtPirin1
transcript levels were examined by relative quantitative RT-PCR in
9-day-old light-grown Arabidopsis plants sprayed with 100 M ABA ()
or distilled water (). The ABA-responsive Rab18 transcript (Lang and
Palva, 1992) served as a positive control for the assay.
(B) Transcript levels of the AtPirin1 gene are induced by red light.
AtPirin1 transcript levels were examined by relative quantitative RT-PCR
in seedlings grown for 6 days in the dark and treated with a mock
pulsed light (d) or irradiated with a single pulse of low-fluence blue light
(b) or low-fluence red light (r).
18S rRNA was used as an endogenous RT-PCR standard. Representative experimental replicates are shown.
mined by RT-PCR product densitometric analysis of AtPirin1
and 18S relative quantitative amplification standards. This result suggests that ABA-inducible cis-acting regulatory elements
in the AtPirin1 promoter region are functional.
When we tested AtPirin1 transcript levels under different light
conditions, we found that a single pulse of low-fluence red light
(104 mol·m2·s1) causes an increase in AtPirin1 transcript
levels compared with those in dark-grown seedlings (Figure
3B). A single pulse of low-fluence blue light did not elicit a
noticeable response (Figure 3B). Densitometric analysis of
AtPirin1 and 18S amplification levels indicated that the pulse of
low-fluence red light leads to an approximately threefold increase in AtPirin1 transcript levels in dark-grown Arabidopsis
seedlings.
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Identification of atpirin1 T-DNA Insertional Mutants
To examine the role of AtPirin1 in plants, we obtained two different mutant lines, atpirin1-1 and atpirin1-2, that carry a T-DNA
insertion in the AtPirin1 gene. The atpirin1-1 mutant harboring a
T-DNA insertion in intron 5 of the AtPirin1 gene (Figure 2B) was
found in a PCR screen of a collection of 60,480 T-DNA–inserted
Arabidopsis lines using an AtPirin1-specific primer and a T-DNA
left border–specific primer (Krysan et al., 1999). The genetic
background for this mutant line is ecotype Wassilewskija (Ws).
The atpirin1-2 mutant harboring a T-DNA insertion in exon 4 of
the AtPirin1 gene (Figure 2B) was obtained from the Salk Institute Genomic Analysis Laboratory. The genetic background for
this mutant line is ecotype Columbia (Col). DNA gel blot and
PCR analyses of atpirin1 mutants and their progeny revealed
that there is only one T-DNA insertion in each of these mutant
lines (data not shown).
No homozygous atpirin1-1 insertional mutants were found
because of the developmental arrest of embryos at the globular
stage of development, whereas the development of heterozygous atpirin1-1 mutant seeds was indistinguishable from that
of the wild type (data not shown). Introduction of the AtPirin1
transgene under a moderate protochlorophyllide oxidoreductase A (PorA) promoter or a constitutive 35S promoter of Cauliflower mosaic virus did not lead to complementation of the
embryo-lethal phenotype in atpirin1-1 mutants. atpirin1-2 homozygous mutants do not display the embryo-lethal phenotype. Together, these data suggest that the atpirin1-1 homozygous lethality probably is caused by a mutation in a linked
gene.
For phenotypic analysis, comparisons were made between
heterozygous atpirin1-1 and heterozygous and homozygous
atpirin1-2 mutant plants as well as between two null alleles of
GPA1, gpa1-1 and gpa1-2. Note that atpirin1-1 and atpirin1-2
seeds, referred to herein as “heterozygous,” are in fact the
progeny of heterozygous plants and, as determined by PCR
analysis, represent an 2:1 mixture of heterozygous and wildtype seeds in the case of atpirin1-1 and an 1:2:1 mixture of
homozygous, heterozygous, and wild-type seeds in the case of
atpirin1-2.
Germination of atpirin1 Mutant Seeds Is Hypersensitive
to ABA
In the presence of 500 to 1000 nM ABA, germination of atpirin1
mutant seeds was delayed (Figures 5A to 5D). An ABAimposed inhibition of gpa1 seed germination was observed recently, although under somewhat different growth conditions
(Ullah et al., 2002). To fully compare the atpirin1 and gpa1 mutant phenotypes, all seeds were harvested, stored, and tested
under identical conditions. Matched seed lots were planted on
half-strength Murashige and Skoog (1962) medium lacking sucrose and Gamborg vitamins, stratified, and germinated at
room temperature under continuous white light. On day 10, almost 80% of all wild-type Col seeds germinated, although only
31% of atpirin1-2 homozygous mutant seeds germinated, in
Germination of atpirin1 Mutant Seeds Is Delayed
in the Absence of Stratification
In the absence of stratification treatment (4
C for 48 h), germination of atpirin1-2 homozygous mutant seeds was delayed
compared with that of wild-type seeds (Figure 4A). There was a
slight delay in the germination of atpirin1-1 (Figure 4B) and
atpirin1-2 (Figure 4A) heterozygous seeds as well. Stratification
brought atpirin1-1 and atpirin1-2 germination to wild-type levels (Figure 4). Importantly, germination of the GPA1 homozygous mutant lines gpa1-1 and gpa1-2 also was delayed somewhat under the conditions tested (Figure 4B). As with atpirin1,
stratification increased gpa1-1 and gpa1-2 germination to wildtype levels (Figure 4B). Germination profiles of the atpirin1 and
gpa1 mutants suggest an increased state of dormancy that is
alleviated by stratification, which is known to be a dormancybreaking factor.
Figure 4. Seed Germination of atpirin1 and gpa1 Mutants.
(A) Germination of wild-type Col and atpirin1-2 mutant seeds.
(B) Germination of wild-type Ws, atpirin1-1, gpa1-1, and gpa1-2 mutant
seeds.
Matched seed lots on premoistened filter paper were placed directly at
22
C under continuous white light for germination. Values presented are
mean percentages of germination from three independent experimental
replicates (each replicate included 70 to 150 seeds per line) with standard errors. Heterozygous and homozygous mutants are indicated by
/ and /, respectively, next to the corresponding mutant genotype. Germination data for stratified wild-type Col seeds (wild type Col
str.) and stratified wild-type Ws seeds (wild type Ws str.) are included in
(A) and (B), respectively. Upon stratification, there was no statistically
significant difference in germination rates between wild-type and corresponding mutant seeds.
AtPirin1/GPA1 Role in Seed Germination
1583
Figure 5. Seed Germination and Early Seedling Development of atpirin1 and gpa1 Mutants Are Hypersensitive to ABA.
Matched seed lots on half-strength Murashige and Skoog (1962) agarose medium supplemented with ABA were stratified for 48 h at 4
C before being
placed at 22
C under continuous white light for germination. Heterozygous and homozygous mutants are indicated by / and /, respectively,
next to the corresponding mutant genotype.
(A) Germination of wild-type Col seeds and atpirin1-2 mutant seeds in the presence of the indicated concentrations of ABA at day 10 after stratification.
(B) Germination of wild-type Ws seeds and atpirin1-1, gpa1-1, and gpa1-2 mutant seeds in the presence of the indicated concentrations of ABA at
day 10 after stratification.
(C) Germination of wild-type Col seeds and atpirin1-2 mutant seeds in the presence of 800 nM ABA over time (days after stratification).
(D) Germination of wild-type Ws seeds and atpirin1-1, gpa1-1, and gpa1-2 mutant seeds in the presence of 800 nM ABA over time (days after stratification).
Values shown in (A) to (D) are mean percentages of germination from three independent experimental replicates (each replicate included 70 to 150
seeds per line) with standard errors.
(E) Early seedling development of atpirin1-2 mutants is inhibited by ABA. No ABA () or 500 nM ABA () was added to the growth medium in
phytatrays. Note that 50% of the mutant seeds shown are germinated as judged by radicle emergence (cf. with [A]).
(F) Early seedling development of atpirin1-1, gpa1-1, and gpa1-2 mutants is inhibited by ABA. No ABA () or 800 nM ABA () was added to the
growth medium in phytatrays. Note that 30 to 40% of the mutant seeds shown are germinated as judged by radicle emergence (cf. with [D]).
Photographs for (E) and (F) were taken on day 10.
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the presence of 800 nM ABA (Figure 5C). Under identical conditions, 50% of the atpirin1-1 heterozygous seeds and 70%
of gpa1-1 and gpa1-2 homozygous mutant seeds failed to germinate by day 10; however, only 31% of wild-type Ws seeds
did not germinate under the same conditions (Figure 5D).
In addition to a reduced rate of seed germination, we found
that ABA also inhibited early seedling development: it significantly delayed the rate of cotyledon expansion and greening in
atpirin1 and gpa1 mutant seedlings. In the presence of exogenous ABA, even when the radicles of many atpirin1 and gpa1
mutant seedlings emerged, the majority of cotyledons failed to
expand and to green (Figures 5E and 5F). Note that under the
growth conditions tested, exogenous ABA inhibited the early
seedling development of atpirin1 and gpa1 mutants to a much
greater extent than it inhibited their germination rate (compare
Figure 5C with 5E and Figure 5D with 5F); therefore, the seedling developmental delay cannot be explained simply by a delay in germination.
We performed a complementation test with atpirin1-1 heterozygous mutants to confirm that the observed ABA-sensitive
phenotype is attributable to the genetic lesion in the AtPirin1
gene. Heterozygous atpirin1-1 plants were transformed with the
AtPirin1 cDNA under the control of the PorA gene promoter. The
PorA promoter is active predominantly in etiolated seedlings,
and PorA mRNA disappears within the first 4 h of greening
(Armstrong et al., 1995; Runge et al., 1996). Figures 6A and 6B
show that the PorA-AtPirin1 transgene was able to complement the ABA-imposed reduction in germination levels of the
atpirin1-1 mutant. Moreover, in one of the complementation
lines, 1-1PorAp2, both ABA-related phenotypes of atpirin1-1
plants (reduced germination and inhibition of early seedling development) were complemented (Figure 6C). This complementation test confirms that the observed atpirin1 mutant phenotypes
result from genetic lesions in the AtPirin1 gene.
In addition to being necessary for the regulation of several
events during seed development, ABA is involved in responses
to environmental stresses, including desiccation and high salt
(Busk and Pages, 1998; Leung and Giraudat, 1998). No difference was noted in atpirin1 seed germination, seedling growth,
or seedling development in the presence of 400 mM mannitol,
200 mM NaCl, or 10% polyethylene glycol. Similarly, mutant
plants displayed no difference in wilting compared with wildtype plants (data not shown). Together, these data suggest that
the role of AtPirin1 with regard to ABA-related processes is limited to seed germination and early seedling development.
Figure 6. Complementation of the atpirin1-1 ABA-Hypersensitive Phenotype in Plants Carrying the AtPirin1 Transgene.
Matched seed lots on half-strength Murashige and Skoog (1962) agarose medium supplemented with ABA were stratified for 48 h at 4
C before being placed at 22
C under continuous white light for germination.
The atpirin1-1 heterozygous mutant genotype is indicated by /.
(A) Germination of two independent complementation lines, 1-1PorAp1
and 1-1PorAp2, carrying AtPirin1 cDNA under the control of the PorA
promoter in the atpirin1-1 mutant background. Germination was tested
in the presence of the indicated concentrations of ABA at day 10 after
stratification. Wild-type Ws seeds and atpirin1-1 mutant seeds were included for comparison.
(B) Germination of two independent complementation lines, 1-1PorAp1
and 1-1PorAp2, wild-type Ws seeds, and atpirin1-1 mutant seeds in the
presence of 800 nM ABA over time (days after stratification).
Values presented in (A) and (B) are mean percentages of germination
from three independent experimental replicates with standard errors.
(C) Early seedling development of the 1-1PorAp1 and 1-1PorAp2 complementation lines. Wild-type (Ws) and atpirin1-1 mutant seeds were included for comparison. No ABA () or 800 nM ABA () was added to
the growth medium in phytatrays.
AtPirin1/GPA1 Role in Seed Germination
1585
Additionally, we tested atpirin1 sensitivity to gibberellic acid
(GA), because GA is known to function as an antagonist of ABA
during seed germination and gpa1 mutant seed germination
appears to be less sensitive to GA (Ullah et al., 2002). However,
under the conditions tested, we observed no statistically significant difference from the wild type in atpirin1 germination and
early seedling development (data not shown).
The Transition from Vegetative to Reproductive Growth
Is Accelerated in atpirin1 Mutants
atpirin1-1 mutant plants initiate primary shoots and start flowering earlier than wild-type plants, such that a few days before
the onset of flowering, atpirin1 mutants produced primary
shoots of significant length, whereas wild-type plants had very
short shoots (Figure 7A). To analyze this phenotype further, we
measured the time needed for the transition from vegetative to
reproductive growth in atpirin1 mutants and wild-type plants.
Time to flowering was determined by counting the number of
days after stratification until the day the first flower opened. Under long-day growth conditions, which are known to induce
flowering in Arabidopsis plants, 50% of atpirin1-1 plants were
flowering in 23 days; however, only 10% of wild-type Ws
plants were flowering at the same time (Figure 7B). atpirin1-2
homozygous plants also displayed a similar flowering phenotype (Figure 7C). No statistically significant difference in flowering time was noted when atpirin1 plants were grown under
short-day conditions (data not shown).
These results support the hypothesis that AtPirin1 functions
as a moderate negative regulator of flowering under long-day
conditions but is not involved in primary shoot and flowering
initiation. gpa1 mutants do not display any alterations in flowering time under the conditions tested (data not shown), suggesting that AtPirin1 is involved in regulating flowering time in a
GPA1-independent manner.
DISCUSSION
It is likely that the Arabidopsis G-subunit, GPA1, integrates
multiple signaling pathways (Assmann, 2002) and therefore interacts with numerous effector and regulatory proteins. To
date, other than the G protein -subunit, no GPA1-interacting
proteins have been reported. We have identified the 31-kD
AtPirin1 protein as a GPA1-interacting partner that, along with
GPA1, plays a defined role in seed germination and early seedling development.
Pirin was isolated initially from mammals through its physical
interaction with the CAAT-box binding transcription factor NFI/
CTF1 (Wendler et al., 1997). Subsequently, pirin was found to
interact with the ankyrin-repeat domain of the proto-oncoprotein Bcl-3, a nucleus-localized member of the IB family of
transcriptional regulators (Dechend et al., 1999). It is well established that G proteins regulate the activity of a number of
transcription factors through various effectors and/or other intermediates (Neves et al., 2002). GPA1 interaction with AtPirin1
may represent an example of a direct connection between G
proteins and transcriptional regulation.
A perfect CAAT-box sequence along with several of the sur-
Figure 7. Bolting and Flowering Time of atpirin1-1 and atpirin1-2 Plants
in Long-Day Growth Conditions.
(A) At day 17, atpirin1-1 plants have longer bolts and start flowering,
whereas the majority of wild-type Ws plants are just initiating bolting.
(B) Results are plotted as a percentage of plants in the wild-type Ws
and atpirin1-1 heterozygous populations that flower on a particular day.
(C) Results are plotted as a percentage of plants in the wild-type Col
and atpirin1-2 homozygous populations that flower on a particular day.
Flowering was scored when the first flower opened. Values presented in
(B) and (C) are mean percentages of germination from three independent experimental replicates (each replicate included 25 to 40 plants per
line) with standard errors. Number of days was counted from the day
when planted seeds were shifted to long-day conditions after stratification. Heterozygous and homozygous mutants are indicated by / and
/, respectively, next to the corresponding mutant genotype.
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rounding bases defines the core element of the low-fluence
blue light response element in the pea Lhcb1*4 promoter (Folta
and Kaufman, 1999). Plant pirin’s potential to interact with a
G-subunit and a CAAT-box factor, both of which are implicated in the low-fluence blue light system activation of Lhcb1*4
(Warpeha et al., 1991; Folta and Kaufman, 1999), suggests a
role in blue light signal transduction in addition to the ABA
pathways identified here.
Two cis-acting elements, an ACGT core and a TT motif, implicated in ABA- and light-mediated transcription regulation are
found in the 5 regulatory region of the AtPirin1 gene. The
ACGT core element is thought to be the target of basic domain/
Leu zipper transcription factors (Menkens et al., 1995; Leung
and Giraudat, 1998). It has been shown that a phytochromeinteracting factor, PIF3, a member of the basic helix-loop-helix
family, also binds an ACGT element in a sequence-specific
manner and regulates the expression of photoresponsive
genes (Martinez-Garcia et al., 2000). AtPirin1 transcript levels
increase in response to a single pulse of low-fluence red light
and exogenous ABA, suggesting that at least some of the potential upstream cis elements are active. These data, along with
the recent finding that overexpression of GPA1 enhances phytochrome-mediated responses during seedling development
(Okamoto et al., 2001), suggest that both AtPirin1 and GPA1
may be involved in the light regulation of seedling development.
Two atpirin1 mutant lines, atpirin1-1 and atpirin1-2, that harbor distinct T-DNA insertions were used in this study. Both mutant lines display a set of very similar phenotypes with regard to
seed germination, ABA sensitivity, and flowering time, indicating that these pleiotropic effects are the result of T-DNA insertions in the AtPirin1 gene. Also, the PorA-Pirin transgene complements the ABA-imposed delay in the germination and early
seedling development of atpirin1-1.
The atpirin1 and gpa1 mutants display a significant reduction
in germination rates in the absence of stratification. When germination rates were tested in the presence of exogenous ABA,
both atpirin1 and gpa1 mutants displayed less than wild-type
germination rates. Furthermore, exogenous ABA inhibited the
expansion and greening of cotyledons in atpirin1 and gpa1 mutant seedlings. A subset of atpirin1 mutant phenotypes correspond to gpa1 mutant phenotypes, confirming that these interacting proteins function in the same pathway.
The reduced germination rates of the atpirin1 and gpa1 mutants likely are the result of the enhanced dormancy of these
mutant seeds, which is further facilitated by the application of
exogenous ABA, a plant hormone known to establish seed dormancy during embryo maturation (Bonetta and McCourt, 1998;
Koornneef et al., 2002). The recent observation that overexpression of the Arabidopsis G protein–coupled receptor GCR1
abolishes seed dormancy (Colucci et al., 2002) further implicates the plant G protein pathway in the regulation of seed dormancy.
The two alleles have similar genetic characteristics with respect to flowering and germination in the absence of cold or
ABA treatment. By contrast, atpirin1-1 and atpirin1-2 display
apparent dominant and recessive natures, respectively, with regard to ABA effects on seed germination and early seedling development. The differences between atpirin1-1 and atpirin1-2
may reflect varietal differences with respect to ABA sensitivity
and/or the maternally based ABA-mediated expression of the
Atpirin1 gene.
atpirin1 and gpa1 are similar to a small group of mutants, including era1, fiery1, abh1, and sad1, that display increased
seed dormancy and ABA hypersensitivity (Cutler et al., 1996;
Hugouvieux et al., 2001; Xiong et al., 2001a, 2001b). These mutations are recessive, indicating that the proteins involved
might be negative regulators of ABA action. Like atpirin1 and
gpa1, these mutations have multiple phenotypes, some without
evident connection to ABA signaling, suggesting that these
proteins may be involved in the regulation of a number of different processes in plants.
It was suggested recently that gpa1 mutant seeds display
lower germination rates in the presence of ABA as a result of
the reduced sensitivity of these mutants to GA (Ullah et al.,
2002). Under the germination conditions used in the present
study (half-strength Murashige and Skoog [1962] medium lacking sucrose and Gamborg vitamins, cold pretreatment, and
germination in continuous white light), mutants of the potential
GPA1 effector, AtPirin1, had increased ABA sensitivity but did
not display a reduction in GA sensitivity, suggesting that ABA
hypersensitivity and GA hyposensitivity during gpa1 seed germination may result from lesions in two separate pathways.
Besides delaying the germination rates of atpirin1 and gpa1
mutants, ABA delays their overall seedling development: the
majority of cotyledons failed to green and expand. Therefore,
even though a significant fraction of mutant seeds germinated
in the presence of low ABA concentrations, as judged by the
full penetration of the seed coat by the root tip (Figures 5A to
5D), the seedlings still were growth arrested (Figures 5E to 5F).
A similar phenotype has been reported for two other ABAhypersensitive mutants, fiery1 and sad1 (Xiong et al., 2001a,
2001b). Previously, it was suggested that ABA inhibits cotyledon greening and cell expansion more strongly than the breakdown of the seed coat (Steber et al., 1998).
In addition to germination-related phenotypes, atpirin1 mutants exhibit a moderate early-flowering phenotype when
grown under long-day conditions. Early-flowering mutant phenotypes are thought to result from the inactivation of genes required to repress flowering (Coupland, 1995). Overexpression
of the putative Arabidopsis G protein–coupled receptor GCR1
accelerates flowering as well as the expression of the flowering-inducing gene LFY (Colucci et al., 2002). However, we observed no difference in flowering time in gpa1 mutants, suggesting that AtPirin1-mediated flowering regulation is a G
protein–independent process. A model illustrating AtPirin1’s involvement in flowering time regulation is presented in Figure 8
(at right).
Based on the AtPirin1 gene characterization and atpirin1
mutant phenotypes, there are two conceivable models for
AtPirin1’s mode of action. The first model postulates that, with
regard to germination and early seedling development, AtPirin1
functions in a negative feedback regulatory loop of ABA signaling and is involved in resetting the ABA signal transduction
pathway (Figure 8). According to the second model, AtPirin1
also relieves the ABA-imposed inhibition of germination, although indirectly, through the activation of germination-pro-
AtPirin1/GPA1 Role in Seed Germination
1587
Yeast and Bacterial Strains
For the yeast two-hybrid experiments, the Saccharomyces cerevisiae
L40 strain was used. The partial genotype of L40 is MATa his3-200 trp1901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-LacZ GAL4
(Vojtek et al., 1993). Escherichia coli strain KC8 (Clontech Laboratories,
Palo Alto, CA), which is auxotrophic for Leu and Trp, was used for plasmid recovery from yeast. E. coli protease-deficient strain BL21 was used
for glutathione S-transferase (GST) fusion protein expression.
Plasmid Construction
Figure 8. Schemes of AtPirin1 Modes of Action.
Together with GPA1, AtPirin1 positively regulates seed germination and
early seedling development by overcoming the negative effects of ABA
and/or activating germination-promoting pathway(s). AtPirin1 mRNA
levels are upregulated by ABA, suggesting the existence of a negative
feedback regulatory loop of the ABA signaling pathway. Independent of
GPA1, AtPirin1 is involved in the regulation of flowering time. Thick arrows indicate either known pathways or pathways established in this
study.
moting processes (Figure 8). These models are not mutually exclusive and, importantly, GPA1 can be envisioned to function
together with AtPirin1 in both of them.
The isolation and characterization of the AtPirin1 protein and
the characterization of its mutant phenotypes provide biochemical and genetic evidence that AtPirin1 is a multifunctional
regulatory protein that likely operates immediately downstream
of its interacting partner, GPA1, in regulating seed germination
and early seedling development.
METHODS
Plant Materials and Growth Conditions
Both Columbia (Col) and Wassilewskija (Ws) ecotypes of Arabidopsis
thaliana were used as wild-type controls for phenotype comparison. For
germination assays, seeds were placed in 15-cm Petri dishes on filter
paper saturated with distilled water and, where specified, stratified (4
C
for 48 h) before being placed at room temperature in continuous white
light (20 mol·m2·s1) for germination. For hormonal assays, stress tolerance assays, and RNA isolation from abscisic acid (ABA)–treated
plants, seeds were planted in phytatrays (Sigma) on half-strength
Murashige and Skoog (1962) 0.8% (w/v) agarose medium lacking sucrose
and Gamborg vitamins, stratified, and germinated as described above.
For flowering tests, plant transformation, and bulking up of seeds, plants
were grown on Metro-Mix 200 growing soil medium (Scotts, Maysville,
OH) under white light supplied by cool-white fluorescent bulbs (90
mol·m2·s1). The atpirin1-1 T-DNA insertion line was isolated by
screening the population of the Arabidopsis T-DNA insertion lines generated in the Ws ecotype at the Arabidopsis Knockout Facility at the University of Wisconsin (Madison). The atpirin1-2 T-DNA insertion line in the
Col ecotype background was obtained from the Salk Institute Genomic
Analysis Laboratory through the ABRC (Columbus, OH).
For the two-hybrid assay, protein fusions to the DNA binding domain of
the bacterial repressor LexA were made using the plasmid pLexA-NLS, a
modified version of the pBTM116 plasmid (Vojtek et al., 1993) containing
nuclear localization signal. The GPA1 cDNA was obtained by PCR from
pCIT1828 plasmid using primers designed to incorporate partial EcoRI
and XhoI restriction sites. The GPA1 bait plasmid was created by cloning
the GPA1 cDNA into the pLexA-NLS plasmid (EcoRI-SalI) after exoIII
(Promega) modification of PCR product ends (Kaluz et al., 1992). For in
vitro protein association assays, the restriction fragment of GPA1 cDNA
(EcoRI-XhoI) was subcloned into the GST fusion expression vector
pGEX-4T-1 (Amersham Pharmacia Biotech). For the complementation
test of the atpirin1-1 mutant, the AtPirin1 cDNA was obtained by reverse
transcription (RT)–PCR using primers designed to incorporate partial
NcoI restriction sites. After exoIII modification of the PCR product ends,
the AtPirin1 cDNA was cloned into the vector pBSK101.4PorA (NcoI), a
modified version of pBSK101.3 (Folta and Kaufman, 1999) in which the
-glucuronidase (GUS; uidA) coding region was replaced with a multiple
cloning site (EcoRI-HindIII-NcoI) and the PorA gene promoter was released from the pAG3/6 plasmid and inserted into HindIII-NcoI sites. The
PorA-AtPirin1 fusion was moved into the plant transformation vector
CLS9600, a derivative of pPZP100 (Hajdukiewicz et al., 1994). CLS9600
contains an additional cassette consisting of the minimal blue light promoter (the 95 to 2 fragment of the PsLhcb1*4 promoter), the GUS
gene, and the nopaline synthase terminator (Folta and Kaufman, 1999).
Yeast Two-Hybrid Screening
The GPA1 bait construct was used to screen the Kim and Theologis
ACT two-hybrid cDNA library (ABRC). Yeast transformations were performed using the lithium acetate/polyethylene glycol–based method
(Gietz and Sugino, 1988). Standard yeast growth medium was prepared as
described in the Yeast Protocol Handbook from Clontech. The S. cerevisiae reporter strain L40 was transformed first with the GPA1 bait vector
and subsequently with the Arabidopsis two-hybrid cDNA library. Transformants were plated on 15-cm synthetic dextrose plates (Trp, Leu,
Lys, His) to select for His prototrophy. The His colonies were restreaked on 5-bromo-4-chloro--D-galactoside–containing selective
medium, and LacZ reporter gene activity was monitored visually. Library
plasmids from HisLacZ yeast colonies were rescued by direct retransformation from LacZ cells into the E. coli strain KC8 according to a
standard protocol. E. coli colonies grown on medium lacking Leu were
selected. Positive clones were sequenced at the Cancer Research Center DNA Sequencing Facility at the University of Chicago.
Expression of the GST-GPA1 Fusion Protein
The GST-GPA1 fusion protein was expressed in E. coli strain BL21. Protein expression was induced with 100 M isopropyl--D-thiogalactopyranoside, and extracts were prepared by treating cells for 3 min at 0
C
with lysozyme solution (1 mg/mL lysozyme and 2.5 mM Tris, pH 8.0) followed by a brief sonication. Recombinant proteins were affinity-purified
1588
The Plant Cell
on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech).
Samples were visualized on SDS-PAGE gels stained with Coomassie
Brilliant Blue G 250.
In Vitro Binding Assay
Expression of the GST-GPA1 fusion proteins was induced as described
above. The radiolabeled AtPirin1 protein was produced by coupled in
vitro transcription/translation using the TNT T7 Coupled Wheat Germ
Extract System (Promega). Translation-grade 35S-Met (Amersham International Redivue) was obtained from Amersham Pharmacia Biotech. A
typical binding reaction was prepared by mixing 20 L of 50% GSTfused protein immobilized on glutathione-Sepharose 4B beads, 300 L
of binding assay buffer (10 mM Tris, pH 7.2, 50 mM potassium acetate,
pH 7.0, 0.01% Triton X-100, and 4 mM magnesium acetate), and 20 L
of AtPirin1 in vitro translation reaction. Binding reactions were agitated
on an orbital shaker for 2 h at room temperature. The beads were pelleted and washed four times with the binding assay buffer. Samples
were resolved on a 10% SDS-PAGE gel, and the resulting gel was fixed
and fluorographically enhanced with EN3HANCE Autoradiography Enhancer (DuPont–New England Nuclear Life Science Products) according to the manufacturer’s instructions and visualized by autoradiography.
In specified cases, the GST-GPA1 fusion was loaded with GTPS
(Sigma) before addition to a binding reaction. GST-GPA1 loading with
GTPS was achieved by incubating 15 L of washed GST-GPA1–
Sepharose beads with 10 L of 10 mM GTPS in 90 L of wash buffer
(50 mM Tris, 100 mM NaCl, 0.1% Triton X-100, and 10 mM magnesium
acetate) and agitating the reaction overnight on an orbital shaker
at 4
C.
Identification of the atpirin1-1 T-DNA Insertion Mutant
The population of 64,000 Arabidopsis T-DNA insertion lines at the
University of Wisconsin Arabidopsis Knockout Facility was screened using pirinTdir primer (5-ATACATGCTACAGGGAGGTATCATTCACA-3)
according to the facility’s protocol (www.biotech.wisc.edu/Arabidopsis/).
The progeny of a plant heterozygous for a T-DNA insertion in the AtPirin1
gene were genotyped by PCR using pirinTdir primer and JL202 T-DNA
left border–specific primer (www.biotech.wisc.edu/Arabidopsis/). As a
result of the embryonic lethality of plants homozygous for a T-DNA insertion in the AtPirin1 gene, heterozygous plants were distinguished by histochemical GUS staining of flowers and selected for propagation. Flowers were chosen for GUS staining because the T-DNA insert vector
contains the GUS reporter gene driven by the flower-specific apetala3
promoter. GUS histochemical staining was conducted essentially as described (Jefferson et al., 1987). In brief, freshly excised flowers were immersed in GUS staining solution containing 8 mM 5-bromo-4-chloro-D-glucuronide cyclohexylammonium salt (Gold BioTechnology, St.
Louis, MO) and 2% Triton X-100 in 50 mM sodium phosphate buffer, pH
7.2, and then incubated at 37
C overnight.
Germination Assay
Germination rates were compared between seed lots that were produced, harvested, and stored under identical conditions. Seventy to 150
seeds from wild-type (Col and Ws), atpirin1-1, gpa1-1, gpa1-2, and
atpirin1-2 plants were planted in triplicate. Seeds were considered germinated when the radicles completely penetrated the seed coat. Germination was scored daily for 10 days after seeds were placed at room
temperature.
Hormonal and Stress Tolerance Assays
Expression Studies by Relative Quantitative RT-PCR
To study the effects of ABA, 9-day-old Arabidopsis seedlings were
sprayed with 100 M ABA or sterile distilled water and plant tissue was
harvested 4 h later. To study the effects of light, planting was performed
as described (Folta and Kaufman, 1999). Six-day-old dark-grown Arabidopsis seedlings were irradiated with a single pulse of low-fluence blue
or red light (104 mol·m2·s1). Plant tissue was harvested 2 h later. Total
RNA was isolated with TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. RNA was used as
a template for first-strand DNA synthesis using the SuperScript FirstStrand Synthesis System for RT-PCR (Invitrogen Life Technologies,
Carlsbad, CA). PCR amplification was performed using Elongase Enzyme Mix (Invitrogen Life Technologies); the optimal concentration of
Mg2 for these amplification reactions was 1.3 mM. PCR amplifications
were performed in a final volume of 23 L with RtpirinD (5-TGACGTATGAGAACAATAGTGTAC-3) and pirinTrev (5-GTAACCTCTAACACTTGGCCATTTCAAC-3), AtPirin1-specific primers that distinguish between
the AtPirin1 transcript and genomic DNA contaminants. Conditions were
chosen in the linear range of amplification for the products and were as
follows: 30 cycles of 94
C for 20 s, 94
C for 15 s, 60
C for 30 s, and 72
C
for 2 min, followed by 72
C for 2 min. QuantumRNA 18S Internal Standards (Ambion, Austin, TX) were used as a relative quantitative amplification control. To achieve levels of amplification comparable to those of
the AtPirin1 PCR products, 18S-specific primers were equilibrated with
18S PCR Competimers in a ratio of 2:8, which corresponds to the abundance levels of rare transcripts. The PCR products were resolved on a
1% agarose gel, and amplification levels were determined using the onedimensional multidensitometry tool on an AlphaImager 2000 (Alpha Innotech, San Leandro, CA).
The effects of ABA on seed germination were studied by determining the
germination rates of 70 to 150 seeds planted in triplicate on growth medium containing ABA (mixed isomers; Sigma). For direct comparisons of
germination rates at a particular ABA concentration, each phytatray was
subdivided and all seed lines were planted on the same phytatray.
The effects of GA on seed germination were determined as described
for ABA except that 1 or 10 M GA (Sigma) was added to the medium.
The effects of mannitol, polyethylene glycol, and NaCl were studied in a
similar manner.
Complementation Analysis
Complementation constructs carrying the AtPirin1 cDNA under the control of the PorA promoter are described above. atpirin1-1 heterozygous
Arabidopsis plants were transformed by vacuum infiltration (Bechtold et
al., 1993). Transgenic plants were selected by resistance to the herbicide
Finale (Roussel Uclaf, Montvale, NJ) and by histochemical GUS staining
of leaves. atpirin1-1 heterozygous plants carrying the PorA-AtPirin1
transgene were selected by PCR.
Flowering Time Analysis
Wild-type and mutant plants for corresponding experimental replicates
were grown in the same tray, which was rotated 180
every 24 h. For
each experimental replicate, 25 to 40 plants were analyzed per line.
Long-day conditions consisted of 16 h of light followed by 8 h of darkness. The onset of flowering was defined as the day when the first flower
opened. atpirin1-1 heterozygous plants were selected by histochemical
GUS staining of flowers as described above.
AtPirin1/GPA1 Role in Seed Germination
Upon request, all novel materials described in this article will be made
available in a timely manner for noncommercial research purposes.
Accession Numbers
Accession numbers for the sequences shown in Figure 2A are as follows:
AtPirin1 (NP_191481), AtPirin2 (NP_191485), AtPirin3 (NP_030851),
AtPirin4 (NP_175474), tomato pirin (TOMpirin; AAF22236), and human
pirin (HUMpirin; CAA69195).
ACKNOWLEDGMENTS
We thank Alan Jones for providing the gpa1-1 and gpa1-2 mutant seed
lines, Stanley Hollenberg for the pLexA-NLS plasmid, Hong Ma for the
pCIT1828 plasmid, Gregory Armstrong for the pAG3/6 plasmid, Caren
Chang for the ETR1 bait construct, and Joseph Kieber for the CTR1 prey
construct. We also thank Mary Beth Anderson, Kevin Folta, and John
Marsh for their assistance in the cultivation and transformation of Arabidopsis and for their useful discussions, Dmitri Pestov and Sanghamitra
Saha for their useful suggestions, and Sam Hawkins for technical assistance. This work was supported by grants from the U.S. Department of
Agriculture and the National Science Foundation to L.S.K.
Received March 14, 2003; accepted April 28, 2003.
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The Arabidopsis Cupin Domain Protein AtPirin1 Interacts with the G Protein α-Subunit GPA1
and Regulates Seed Germination and Early Seedling Development
Yevgeniya R. Lapik and Lon S. Kaufman
Plant Cell 2003;15;1578-1590; originally published online June 5, 2003;
DOI 10.1105/tpc.011890
This information is current as of June 17, 2017
References
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