First published online June 12, 2003; 10.1104/pp.102.019141
Plant Physiology 132:1560-1576 (2003)
© 2003 American Society of Plant Biologists
DEVELOPMENT AND HORMONE ACTION
Abscisic Acid and Gibberellin Differentially Regulate Expression of Genes of the SNF1-Related Kinase Complex in Tomato Seeds1
Kent J. Bradford*,
A. Bruce Downie3,
Oliver H. Gee2,
Veria Alvarado4,
Hong Yang5 and
Peetambar Dahal
Department of Vegetable Crops, One Shields Avenue, University of
California, Davis, California 95616–8631
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ABSTRACT
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The SNF1/AMP-activated protein kinase subfamily plays central roles in
metabolic and transcriptional responses to nutritional or environmental
stresses. In yeast (Saccharomyces cerevisiae) and mammals, activating
and anchoring subunits associate with and regulate the activity, substrate
specificity, and cellular localization of the kinase subunit in response to
changing nutrient sources or energy demands, and homologous SNF1-related
kinase (SnRK1) proteins are present in plants. We isolated cDNAs corresponding
to the kinase (LeSNF1), regulatory (LeSNF4), and
localization (LeSIP1 and LeGAL83) subunits of the SnRK1
complex from tomato (Lycopersicon esculentum Mill.). LeSNF1 and
LeSNF4 complemented yeast snf1 and snf4 mutants and
physically interacted with each other and with LeSIP1 in a glucose-dependent
manner in yeast two-hybrid assays. LeSNF4 mRNA became abundant at
maximum dry weight accumulation during seed development and remained high when
radicle protrusion was blocked by abscisic acid (ABA), water stress, far-red
light, or dormancy, but was low or undetected in seeds that had completed
germination or in gibberellin (GA)-deficient seeds stimulated to germinate by
GA. In leaves, LeSNF4 was induced in response to ABA or dehydration.
In contrast, LeSNF1 and LeGAL83 genes were essentially
constitutively expressed in both seeds and leaves regardless of the
developmental, hormonal, or environmental conditions. Regulation of
LeSNF4 expression by ABA and GA provides a potential link between
hormonal and sugar-sensing pathways controlling seed development, dormancy,
and germination.
The transition between seed development and germination is accompanied by
large-scale changes in gene expression patterns and metabolic pathways. During
seed maturation, enzymes and pathways involved in storage reserve accumulation
and preparation for desiccation predominate
(Girke et al., 2000 ). Upon
imbibition after seed maturation and desiccation, different sets of genes are
expressed for reserve mobilization, tissue weakening, and embryo expansion
associated with radicle protrusion and seedling growth
(Bradford et al., 2000;
Gallardo et al., 2002).
Abscisic acid (ABA) is an important regulator of seed development and
maturation, and several transcription factors determining ABA sensitivity,
including ABI3/VP1, ABI4, and ABI5, along with biosynthetic enzymes
controlling ABA amount (ABA1, ABA2, and ABA3), are involved in promoting
reserve accumulation, developmental arrest, and the imposition of dormancy
(Finkelstein et al., 2002;
Koornneef et al., 2002). GAs,
on the other hand, are essential for germination and subsequent reserve
mobilization to support seedling growth
(Karssen, 1995;
Debeaujon and Koornneef,
2000). Antagonistic interactions between ABA and GA in regulating
reserve mobilization in cereal aleurone layers are well documented
(Gomez-Cadenas et al.,
2001).
Recently, screens for mutants altered in their sensitivity to the presence
of sugars during germination and early seedling development have revealed
interactions between sugar and hormonal signaling pathways
(Finkelstein and Gibson, 2001;
Rolland et al., 2002). In
particular, a series of mutations have been identified in Arabidopsis based on
the ability of mutant seeds to complete germination and of seedlings to
develop in the presence of concentrations of Glc, Man, or Suc that are
inhibitory to wild-type seeds. These include mig (Man-insensitive
germination), sun (Suc-uncoupled), sis (sugar-insensitive),
gin (Glc-insensitive), isi (impaired Suc induction), and
other mutants exhibiting altered germination or seedling growth responses to
sugars (for review, see Finkelstein et
al., 2002). Interestingly, a number of mutations identified in
this way are allelic to previously known mutations in ABA synthesis
(aba) or sensitivity (abi). For example, sun6, sis5,
gin6, and isi3 are allelic to abi4; sis4,
gin1, and isi4 are allelic to aba2; and some (abi4,
abi5, aba1, aba2, and aba3), but not all (abi1, abi2,
and abi3), aba and abi mutants exhibit
sugar-insensitive phenotypes (Finkelstein
et al., 2002). Furthermore, provision of additional sugars
partially overcame the inhibition of germination by ABA in both wild-type and
abi mutant seeds (Garciarrubio et
al., 1997; Finkelstein and
Lynch, 2000a). Conversely, ABA increased the expression of a
starch biosynthetic gene (ApL3) in response to sugars, suggesting
that ABA can enhance the ability of tissues to respond to sugar signals
(Rook et al., 2001). ABA also
plays important but distinct roles in sugar, osmotic, and cold stress
signaling during germination and early seedling growth
(Cheng et al., 2002;
Kim et al., 2003).
Interactions between sugar and hormonal signaling pathways have also been
reported for GA, ethylene, and cytokinins (for review, see
Gazzarrini and McCourt, 2001;
Rolland et al., 2002). Thus,
hormonal and sugar regulation of gene expression and plant function are
intimately linked, particularly in the case of the transition from seed
maturation to germination.
The mechanisms by which plant cells sense sugars and regulate carbohydrate
metabolism are complex and multiple pathways have been identified
(Gibson, 2000;
Smeekens, 2000;
Rolland et al., 2002). The
SNF1-related kinase (SnRK1) complex is thought to be a central component of
the sugar sensing and response mechanism
(Halford and Hardie, 1998;
Halford et al., 2000,
2003). First identified
genetically in yeast (Saccharomyces cerevisiae) as mutants incapable
of derepressing Glc-regulated genes and, therefore, unable to grow on sugars
other than Glc, the SNF1 (Suc non-fermenting 1) kinase complex and the related
mammalian AMP-activated protein kinase (AMPK) complex are metabolic sensors of
Glc availability and AMP to ATP ratios, respectively
(Hardie et al., 1998). Protein
sequence and functional homologies exist between the yeast and mammalian
kinase ( ) subunits (SNF1/AMPK), regulatory ( ) subunits
(SNF4/AMPK), and specification/localization ( ) subunits
(SIP1/SIP2/GAL83/AMPK) that constitute the functional kinase complexes.
In yeast, Glc regulates the protein-protein interactions, substrate
specificity, and subcellular localization of this complex that modulate SNF1
kinase activity, resulting in the phosphorylation of activators and repressors
that control transcription of multiple genes in metabolic pathways required
for the utilization of alternative energy sources
(Carlson, 1998;
Schmidt and McCartney, 2000;
Vincent et al., 2001). In
mammals, activation of AMPK due to increases in the AMP to ATP ratio during
metabolic stress results in the enhancement of ATP-producing pathways and the
inhibition of ATP-consuming pathways (Kemp
et al., 1999).
Members of several subfamilies of SnRKs have been identified in diverse
plants (Halford et al., 2000,
2003). Those most closely
related to SNF1 (SnRK1 family) exhibit kinase activity on substrates common to
the mammalian and yeast kinases and complement yeast snf1 mutants
(e.g. Alderson et al., 1991;
Muranaka et al., 1994;
Sugden et al., 1999).
Antisense suppression experiments in potato (Solanum tuberosum) and
wheat (Triticum aestivum) indicated that SnRK1s are involved in
regulation of carbon metabolism in planta
(Purcell et al., 1998;
Laurie et al., 2003).
Candidate SNF4-related genes have been identified, including Pv42 in
bean (Phaseolus vulgaris; Abe et
al., 1995) and AKIN
(Bouly et al., 1999) in
Arabidopsis. Plant genes having predicted sequence homology to SnRK1-
subunits are also known, including StubGAL83 from potato
(Lakatos et al., 1999) and
AKIN1 and AKIN2 from
Arabidopsis (Bouly et al.,
1999). Genes in maize (Zea mays;
ZmAKIN-1) and Arabidopsis (AtSNF4 or
AKIN) contain domains homologous to both the - and
-subunits of the SnRK1 complex
(Lumbreras et al., 2001).
Thus, plants contain homologs of all three components common to the yeast and
mammalian SNF1/AMPK complexes, and these components interact in a manner
consistent with a heterotrimeric structure (or a dimeric structure in the case
of the -type proteins) based on yeast two-hybrid and in vivo
protein interaction studies (Bouly et al.,
1999; Lakatos et al.,
1999; Kleinow et al.,
2000; Crawford et al.,
2001; Ferrando et al.,
2001; Lumbreras et al.,
2001). Distinct regulatory components such as PRL1 may mediate
additional functions of SnRK1 kinases in vivo
(Bhalerao et al., 1999;
Farrás et al.,
2001).
In experiments to identify genes expressed or repressed in association with
tomato (Lycopersicon esculentum Mill.) seed germination, we isolated
a cDNA having sequence homology to the -subunit of the SnRK1 complex
(LeSNF4). We report here the functional characterization of this gene
and of tomato SnRK1- and SnRK1- homolog genes in yeast and their
differential expression patterns during tomato seed development and
germination. In addition, we show that expression of only LeSNF4 is
responsive to GA, ABA, and water stress in tomato seeds and leaves. Our
results suggest that hormonal regulation of expression of the -subunit
of the SnRK1 complex could be one mechanism for cross talk between
sugar-sensing pathways and hormonally and environmentally regulated pathways
during seed development and germination and plant responses to stress
conditions.
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RESULTS
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Isolation of Tomato SnRK1 Complex Genes
Differential cDNA display using GA-deficient mutant (gib-1) tomato
seeds imbibed in the presence or absence of GA was employed to identify
candidate gene(s) involved in the regulation or mechanism of seed germination
(Cooley et al., 1999). An mRNA
identified by one of the cDNAs was present in mature gib-1 seeds and
remained abundant in gib-1 seeds imbibed in water (which do not
complete germination), but declined within 12 h in the presence of GA, which
stimulated germination (expression data presented later). A full-length cDNA
corresponding to this mRNA was isolated from a tomato seed cDNA library
prepared from gib-1 seeds imbibed for 24 h in water. The complete
cDNA (GenBank accession no. AF143742) encoded a predicted protein of 373 amino
acids (41,319 kD) having highest sequence homology to a predicted protein from
bean (Pv42; 56% identity, 71% similarity) and a predicted protein from the
Arabidopsis genome (AAD39660; 55% identity, 70% similarity;
Fig. 1A). Regions of homology
were also present in yeast SNF4 protein (21% identity, 40% similarity), in
other Arabidopsis SnRK1- proteins (AKIN and AtSNF4), and in the
-domain of the maize ZmAKIN-1 protein. The LeSNF4, Pv42,
and AAD39660 sequences clustered together, as did the AtSNF4,
ZmAKIN-1, and SNF4 sequences, whereas the AKIN sequence
was divergent from the rest (Fig.
1B). A motif initially identified in the
cystathionine--synthase protein (CBS motif) that is repeated four times
in the yeast SNF4 and mammalian AMPK proteins
(Bateman, 1997;
Kemp et al., 1999) also
appeared four times in the tomato sequence
(Fig. 1A). Sequence
conservation was greatest in the C-terminal regions of the proteins,
coinciding with two of the CBS domains. Based on the sequence homology and
functional complementation (see below), we termed the tomato gene
LeSNF4.
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Figure 1. LeSNF4 encodes a -subunit homolog of the SnRK1 complex. A, Predicted
amino acid sequences of tomato LeSNF4 (AF143742), bean Pv42 (U40713),
Arabidopsis predicted protein (AAD39660), AKIN (P80385), AtSNF4
(AF250335), the -domain (amino acids 125–497) of maize
ZmAKIN-1 (AF276085), and yeast SNF4 (172636) were aligned using
the Clustal method with PAM250 residue weight (DNASTAR Inc., Madison, WI).
Shading indicates extent of conservation/similarity. In addition to direct
sequence homology, CBS motifs based upon protein structure
(http://www.sanger.ac.uk/cgi-bin/Pfam/)
are shared between SNF4 (underlined) and LeSNF4 (overlined). B, Phylogenetic
tree of protein sequences shown in A based upon Clustal analysis (DNAStar
Inc.).
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Hybridization analysis of tomato genomic DNA using LeSNF4 as probe
revealed only single strong bands, indicating that the mRNA is likely to be
encoded by a single gene (Fig.
2A). Genetic mapping supported this conclusion because Chen and
Foolad (1999) used
LeSNF4 as a DNA hybridization marker and mapped it to a single locus
(CG43) on chromosome 6 of the tomato genome. A genomic DNA fragment was
isolated encoding LeSNF4 (termed gLeSNF4; AF419320) that
contained 1,234 bp 5' to the translation start site and two introns of
323 bp (starting at bp 43 from the initial ATG of the open reading frame) and
923 bp (starting at bp 793; data not shown).
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Figure 2. DNA gel-blot analysis of tomato genomic DNA. A, Tomato genomic DNA
(10 µg) was restriction digested with XbaI (X), EcoRI
(E), or BamHI (B), separated on an agarose gel, and hybridized with
LeSNF4 cDNA. B, As above, hybridized with LeSNF1 cDNA. C, As
above, hybridized with LeSIP1 cDNA. D, As above, hybridized with
StubGAL83 cDNA. In a separate experiment using the same restriction
enzymes, hybridization with StubGAL83 or LeGAL83 cDNAs
resulted in identical banding patterns (data not shown).
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A cDNA encoding the tobacco (Nicotiana tabacum) SnRK1-
NPK5 (D26602; Muranaka et al.,
1994) was used to isolate a homologous cDNA (termed
LeSNF1; AF143743) from a tomato seed cDNA library (data not shown).
The predicted amino acid sequence encoded a protein of 514 amino acids (58,824
kD) that is closely related to potato StubSNF1 (U83797; 95% similarity;
Lakatos et al., 1999), NPK5
(87% similarity), and kinases from other species in the SnRK1a group (>80%
amino acid similarity; Halford et al.,
2000). The entire protein sequence of LeSNF1 shared 38% and 43%
similarity with yeast SNF1 (M13971) and rat (Rattus norvegicus)
AMPK (U40819), respectively. The kinase domains comprising the
N-terminal one-half of the protein shared 97% similarity among these plant
proteins and >60% similarity among the plant, yeast, and mammalian
proteins. In the more variable C-terminal region, LeSNF1 shared 92% and 76%
similarity with StubSNF1 and NPK5, but only 12% to 16% similarity with SNF1
and AMPK. Pien et al.
(2001) reported a partial
tomato cDNA encoding 161 C-terminal amino acids having 92% similarity with the
corresponding region of StubSNF1, which they also termed LeSNF1. The
reported sequence is identical with our sequence over this region. Based on
DNA gel-blot analysis, LeSNF1 may be a member of a small gene family
in the tomato genome (Fig.
2B).
A tomato expressed sequence tag (EST; AI486580) having significant homology
to known SnRK1- subunits, or SNF1-interacting proteins (SIPs), was used
to isolate a full-length cDNA (termed LeSIP1; AF322108) from a tomato
seed cDNA library. The predicted amino acid sequence of LeSIP1 shared 50% and
53% amino acid sequence similarity with AKIN1 and StubGAL83
(Fig. 3A) and 70% with
AKIN2 (not shown). LeSIP1, StubGAL83, and AKIN1 fell into a
phylogenetic group distinct from the yeast (SIP2 and GAL83) and the mammalian
(AMPK1) proteins (Fig.
3B). The predicted LeSIP1 amino acid sequence contained a
conserved internal kinase-interacting sequence (KIS) domain and a C-terminal
association with the SNF1 kinase complex (ASC) domain
(Fig. 3A). These domains have
been shown in yeast to interact with SNF1 (-subunit) and with SNF4
(-subunit), respectively (Jiang and
Carlson, 1997). The plant and mammalian proteins are considerably
smaller than the yeast GAL83 and SIP2 proteins, with deletions in the region
between the KIS and ASC domains and in the N-terminal region
(Fig. 3A). DNA gel-blot
analysis of tomato genomic DNA using LeSIP1 as a probe detected one
major band and several weaker bands, suggesting a small family of related
genes in tomato (Fig. 2C). This
was confirmed by probing tomato genomic DNA with StubGAL83, which
resulted in specific hybridization with a restriction fragment pattern
distinct from that of LeSIP1 (Fig.
2D). Little cross hybridization was detected between the two
probes at similar DNA fragment sizes, indicating the specificity of the probes
for the respective genes. The tomato genome apparently contains at least two
SIP-like sequences, LeSIP1 and a tomato homolog of
StubGAL83. We subsequently recovered by reverse transcription-PCR a
partial tomato cDNA sharing 98% identity in both nucleotide and amino acid
sequences with the potato StubGAL83 cDNA
(Fig. 3). A Southern blot of
tomato genomic DNA using this tomato cDNA as a probe exhibited a hybridization
pattern identical to that found when StubGAL83 was used as the probe
(data not shown; Fig. 2D).
Although not full length (230 amino acids versus 289), this tomato cDNA is
evidently the tomato homolog of StubGAL83, so we have termed it
LeGAL83 (accession no. AY245177).
LeSNF4 and LeSNF1 Encode Functional Proteins
Whether LeSNF4 and LeSNF1 encode functional tomato
homologs of the corresponding yeast genes was tested by complementation of
mutants deficient in these genes. In yeast, the SNF4 protein is required for
activation of SNF1 kinase, which is essential for derepression of
sugar-metabolizing enzymes in the absence of Glc. As a consequence,
snf4 mutants are able to grow on Glc but not on Suc or other
carbohydrate sources (Fig. 4A).
Expression of LeSNF4 in a yeast strain having a deletion in the
SNF4 gene (snf4- 2) restored growth on Suc
(or on 2% [w/v] each of Gal, glycerol, and ethanol plus 0.05% [w/v] Glc)
essentially equivalent to that in the mutant yeast complemented with the
wild-type SNF4 gene (Fig.
4A). Complementation was confirmed by measuring invertase activity
from yeast cells in either repressed (2% [w/v] Glc) or derepressed (0.05%
[w/v] Glc) conditions (Fig.
5A). Wild-type yeast exhibited an 8-fold increase in invertase
activity under derepressed conditions. In contrast, only a 50% increase in
invertase activity was detected under derepressed conditions in the
snf4-2 mutant transformed with the empty vector.
However, when snf4-2 yeast cells were transformed to
express either SNF4 or LeSNF4 genes, invertase activity
increased by 14- and 13-fold, respectively, under derepressed conditions
(Fig. 5A). Thus, LeSNF4 is a
functional homolog of SNF4 in regulating derepression of invertase in
yeast.
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Figure 4. Complementation of yeast snf4-2 and
snf1-10 mutants by LeSNF4 and
LeSNF1.A, The snf4-2 mutant was transformed
with the empty vector (sector 1), with yeast SNF4 (sector 3), tomato
LeSNF4 (sector 4), tomato LeSNF1 (sector 5), or yeast
SNF1 (sector 6). Wild-type yeast transformed with the empty vector is
shown in sector 2. Transformed strains containing gene inserts were initially
identified on selective synthetic complete (SC) medium containing Suc (except
those containing empty vector, which were grown on Glc), then were grown on
selective SC plates with 2% (w/v) Glc (left) or Suc (right) for 10 d at
29°C. B, The snf1-10 mutant was transformed with
the empty vector (sector 1) or with tomato LeSNF1 (sector 2) or yeast
SNF1 (sector 3). Transformed strains were selected as above, then
were grown on selective SC plates with 2% (w/v) Glc (left) or Suc (right) for
3 d at 29°C. No colonies were recovered on Suc plates after transformation
of snf1-10 with vectors containing either
LeSNF4 or SNF4 (data not shown). For both A and B, results
similar to those shown on Suc medium were obtained when strains were grown on
medium containing 2% (w/v) each of Gal, glycerol, and ethanol plus 0.05% (w/v)
Glc (data not shown).
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Figure 5. Invertase activity of yeast snf4-2 and
snf1-10 mutants expressing LeSNF4 and
LeSNF1. A, Invertase activity from wild-type and
snf4-2 mutant yeast cells under either repressed (R,
2% [w/v] Glc) or derepressed (D, 0.05% [w/v] Glc) conditions. Mutant cells had
been transformed with the empty vector or vectors expressing SNF4,
LeSNF4, SNF1, or LeSNF1. Error bars indicate
±95% confidence intervals around the means for assays of three to four
independent colonies. B, Invertase activity from
snf1-10 mutant yeast cells under either repressed (R,
2% [w/v] Glc) or derepressed (D, 0.05% [w/v] Glc) conditions. Mutant cells had
been transformed with the empty vector, SNF1, or LeSNF1.
Error bars = ± 95% confidence intervals around the means for assays of
three to four independent colonies.
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Similar experiments were conducted to test whether LeSNF1 encodes
a functional homolog of the yeast SNF1 kinase. The yeast
snf1-10 deletion mutant can grow on Glc but not on
Suc (Fig. 4B). Expression of
LeSNF1 suppressed the mutation and allowed growth on Suc, as did
expression of SNF1 (Fig.
4B). In addition, expression of either SNF1 or
LeSNF1 stimulated secretion of invertase by
snf1-10 cells under both repressed and derepressed
conditions, but invertase activity was greater in low Glc
(Fig. 5B). When LeSNF4
was expressed in snf1-10 cells, no transformants
could be recovered from Suc plates, as expected from the inability of SNF4 to
substitute for SNF1 kinase activity
(Celenza et al., 1989). On the
other hand, expression of either SNF1 or LeSNF1 in
snf4-2 mutant yeast cells restored the ability to
grow on Suc (Fig. 4A) and
stimulated invertase secretion under both repressed and derepressed conditions
(Fig. 5A;
Jiang and Carlson, 1996).
Thus, we conclude that LeSNF1 encodes a functional homolog of SNF1.
LeSNF1, LeSNF4, and LeSIP1 Interact in Yeast Two-Hybrid Assays
The presence of conserved KIS and ASC domains predicted that LeSIP1 would
bind to LeSNF1 and LeSNF4. Therefore, we tested whether the tomato components
of the SnRK1 complex interacted with each other using the yeast two-hybrid
assay (Table I). As expected
(Jiang and Carlson, 1997),
yeast SNF4 and SNF1 proteins showed an interaction that was enhanced under
derepressing (0.05% [w/v] Glc) conditions. LeSNF1 and LeSNF4 proteins
exhibited little interaction in high (2% [w/v]) Glc, but low Glc enhanced
binding by 19-fold. LeSIP1 interacted strongly with both LeSNF1 and LeSNF4
even in high Glc, and this interaction was enhanced approximately 8-fold under
low-Glc conditions. LeSNF1 and LeSNF4 showed little binding activity with SNF4
and SNF1, respectively, in high Glc, but strong interactions occurred in low
Glc. LeSIP1 showed some binding to SNF1 and SNF4 but much less than with the
corresponding tomato proteins. Both LeSNF1 and LeSIP1 exhibited apparent
interaction with themselves under low-Glc conditions. We attempted to test the
ability of StubGAL83 to interact with LeSNF1 and LeSNF4, but high expression
was detected in controls when StubGAL83 was expressed alone in either the
binding or activation domains, precluding meaningful tests (data not
shown).
Differential Expression of SnRK1-Related Genes in Tomato
As noted earlier, LeSNF4 was identified as an mRNA that was
present in mature gib-1 seeds but declined in the presence of GA
(Fig. 6). The expression
pattern was actually more complex because LeSNF4 mRNA first declined
somewhat in gib-1 seeds imbibed in water before increasing to high
abundance after extended imbibition without completing germination
(Fig. 6). When imbibed in 100
µM GA4+7, on the other hand, an
increased abundance of LeSNF4 mRNA was detected after 1 h, but the
amounts then declined to low or undetectable levels before radicle emergence
began at approximately 48 h (Fig.
6). The expression pattern was similar in the endosperm cap
(endosperm tissue enclosing the radicle tip), the radicle tip, and the rest of
the seed (including both the remainder of the embryo and the lateral endosperm
surrounding it; Fig. 6; tissue
print data not shown). Expression was apparently specific to the seed because
the mRNA was not detected in flowers, leaves, or roots of wild-type
field-grown plants (Fig.
6).
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Figure 6. Expression of LeSNF4 mRNA in gib-1 tomato seeds and
wild-type tissues. Seeds were imbibed for the indicated time in either water
or 100 µM GA4+7 at 25°C
before dissecting into the endosperm caps (endosperm tissue covering the
radicle tip), radicle tips, and the remainder of the seed (for diagram, see
Cooley et al., 1999). In
addition, flower (F), leaf (L), and root (R) tissues were collected from
field-grown wild-type (cv Moneymaker) plants. Extracted total RNAs were
hybridized to riboprobes prepared from LeSNF4 (upper) or from a cDNA
fragment (G46) having homology to constitutively expressed ribosomal
protein sequences to confirm equal RNA loading of each lane (lower).
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Because LeSNF4 mRNA was present in mature dry seeds and became
less abundant during germination in response to GA, we examined the expression
of tomato SnRK1-related genes during both seed development and germination.
Tomato seeds initially increased in fresh weight as the tissues expanded,
followed by dry weight accumulation due to deposition of storage reserves
(Fig. 7A). Fresh weight of
extracted seeds then fell, apparently associated with collapse of the testa
cells (Fig. 7A;
Berry and Bewley, 1991). Seed
water content decreased during development, reaching values of 40% to 50%
(w/w, fresh weight basis) at maturity inside the ripe fruit
(Fig. 7A), followed by drying
to 6% to 8% once removed from the fruit. LeSNF4 mRNA was barely
detectable in tomato seeds at 20 and 25 DAF but increased in abundance after
30 DAF and was most abundant in mature seeds (60 DAF;
Fig. 7, B and C). In contrast,
LeSNF1 mRNA was abundant at 20 DAP, then declined somewhat before
increasing again late in seed development
(Fig. 7, B and C).
LeSIP1 mRNA could not be detected in northern analyses of developing
or germinating seeds (data not shown). However, StubGAL83 hybridized
with an mRNA present late in seed development, indicating the expression of
the highly homologous LeGAL83 gene
(Fig. 7, B and C). Therefore,
we used probe prepared from StubGAL83 to assay for the presence of
tomato LeGAL83 mRNA. (These experiments were conducted before the
isolation of the partial LeGAL83 cDNA described above, which
confirmed the virtual identity of their sequences).
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Figure 7. Expression of SnRK1-related genes during tomato seed development.
A, Fresh and dry weights and water contents (fresh weight basis) of tomato
seeds during development. B, Abundance of LeSNF4, LeSNF1,
and LeGAL83 mRNAs during tomato seed development. Total RNA was
extracted from freshly harvested seeds at the times indicated and hybridized
with probes prepared from the different genes (StubGAL83 probe was
used to detect LeGAL83). Lower panels in each pair labeled rRNA are
ethidium bromide-stained gels showing loading of RNA in each lane. C, Relative
mRNA abundance was quantified by densitometry from the images in B and
normalized for RNA loading in each lane. Within each panel, the normalized
signal intensity at 60 d after flowering (DAF) was set equal to 1, and the
normalized signal intensities in other lanes are shown relative to it.
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LeSNF4 mRNA was abundant in mature dry wild-type seeds but
declined in seeds that had not completed germination by 48 h and was not
detected in seeds that had completed germination
(Fig. 8, lanes 1–3).
LeSNF1 and LeGAL83 transcripts were also present in dry
seeds and in imbibed germinating seeds but persisted even after the radicle
had protruded (Fig. 8, lanes
1–3). Imbibing seeds in ABA (100 µM), in an osmotic
solution (–1.3 MPa PEG), or under FR, all of which inhibited radicle
emergence, maintained LeSNF4 mRNA abundance
(Fig. 8, lanes 4–6).
LeSNF4 mRNA remained abundant in naturally dormant seeds that had not
completed germination after 14 d in water
(Fig. 8, lane 7). When these
dormant seeds were transferred to GA, LeSNF4 mRNA decreased but was
still present in seeds that had not completed germination after 48 h and was
absent in germinated seeds (Fig.
8, lanes 8 and 9). In contrast, ABA, osmoticum, dormancy, GA, or
germination status had relatively little effect on the abundance of
LeSNF1 or LeGAL83 mRNAs
(Fig. 8, lanes 1–9).
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Figure 8. Expression of SnRK1-related genes in dry and imbibed tomato seeds
under different conditions. A, Total RNA was extracted from mature dry
wild-type (cv Moneymaker) seeds (lane 1) and seeds imbibed at 25°C under
various conditions (lanes 2–9) and hybridized with probes from
LeSNF4, LeSNF1, and StubGAL83 (to detect
LeGAL83 mRNA). After imbibition in water for 48 h, seeds were
separated into ungerminated (lane 2) and germinated (lane 3) seeds. Additional
seeds were imbibed in 100 µM ABA (lane 4) or in –1.3 MPa
polyethylene glycol (PEG) solution (lane 5) or in water under far-red (FR)
irradiation (lane 6); these conditions prevented the completion of
germination. Naturally dormant seeds were sampled after failing to complete
germination after 14 d of imbibition on water (lane 7). These dormant seeds
were transferred to 100 µM
GA4+7 to stimulate germination, and after 48
h (approximately 50% germination), seeds were separated into ungerminated
(lane 8) and germinated (lane 9) fractions. rRNA, Ethidium bromide-stained gel
showing loading of RNA in each lane. Replicate gels were run with identical
samples and hybridized with RNA probes for LeSNF4, LeSNF1,
or StubGAL83. B, Relative mRNA abundance was quantified by
densitometry from the images in A and normalized for RNA loading in each lane.
Within each panel, the normalized signal intensity of dry seeds (lane 1) was
set equal to 1, and the normalized signal intensities in other lanes are shown
relative to it.
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Because LeSNF4 mRNA was present in mature dry seeds, its continued
presence under conditions preventing germination could be due to increased
stability or reduced turnover rather than continued synthesis. Because its
abundance declined in imbibed seeds before radicle emergence, we tested
whether the transcript would accumulate again if germination were blocked.
When wild-type seeds were imbibed for 36 h in water, LeSNF4 mRNA
amounts decreased before radicle protrusion (e.g.
Fig. 8, lanes 1 and 2). When
these seeds were then transferred to –1.3 MPa PEG or to 4°C,
LeSNF4 mRNA abundance increased, but LeSNF1 and
LeGAL83 mRNA amounts were relatively unaffected (data not shown).
Thus, conditions inhibitory to germination re-induced accumulation after
LeSNF4 mRNA amounts had initially declined.
Although LeSNF4 mRNA was not detected in leaves or roots from
plants grown under normal field conditions
(Fig. 6), we examined the
expression of LeSNF4, LeSNF1, and LeGAL83 in leaves exposed
to ABA or subjected to water stress (Fig.
9). LeSNF4 mRNA was initially low in leaves from
well-watered intact plants (Fig.
9, lanes 1 and 7), in agreement with earlier results
(Fig. 6). Excising leaves and
maintaining them at 100% RWC for 6 h also failed to induce LeSNF4
expression, but LeSNF4 mRNA accumulated in leaves allowed to
dehydrate after excision, with lower RWC resulting in greater accumulation
(Fig. 9, lanes 2–4).
Similarly, excised shoots maintained with their stems in water did not
accumulate LeSNF4 mRNA, but when ABA solution was fed through the
stems, LeSNF4 mRNA increased within 6 h
(Fig. 9, lanes 5 and 6). A
single foliar spray of ABA applied to intact plants also induced transient
accumulation of LeSNF4 mRNA within 6 h, followed by its decline to
undetectable amounts within 24 h (Fig.
9, lanes 7–10). In contrast, comparatively minor changes in
the abundance of LeSNF1 or LeGAL83 mRNAs occurred in shoot
tissues in response to these treatments, although LeSNF1 mRNA
abundance doubled under mild water stress
(Fig. 9, lanes 1–10).
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Figure 9. Expression of SnRK1-related genes in tomato seedling leaves in
response to water stress or ABA. A, Leaves were sampled from well-watered
plants and frozen immediately (lane 1) or were excised and placed in plastic
bags containing moist filter paper either immediately (lane 2) or after
different durations of initial water loss resulting in 90% (lane 3) or 80%
(lane 4) relative water content (RWC). RNA was extracted from the leaves 6 h
after excision. Excised shoots were placed with their cut ends in water (lane
5) or in 100 µM ABA (lane 6) for 6 h before the leaves were
sampled for RNA. Intact seedlings (lane 7 = initial leaf samples) were sprayed
once with 100 µM ABA and leaves were sampled at 6 (lane 8), 12
(lane 9), and 24 h (lane 10) after treatment. rRNA, Ethidium bromide-stained
gel showing loading of RNA in each lane. Replicate gels were run with
identical samples and hybridized with RNA probes for LeSNF4,
LeSNF1, or StubGAL83 (to detect LeGAL83 mRNA). B,
Relative mRNA abundance was quantified by densitometry from the images in A
and normalized for RNA loading in each lane. Within each panel, the normalized
signal intensity of control leaves (lane 1) was set equal to 1, and the
normalized signal intensities in other lanes are shown relative to it.
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DISCUSSION
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Functional SnRK1-Related Genes Are Present in Tomato
Plant enzymatic or -subunits of SnRKs have been grouped into three
subfamilies (SnRK1, SnRK2, and SnRK3) based upon amino acid sequence
homologies (Halford et al.,
2000,
2003). We isolated an
SnRK1-type cDNA, termed LeSNF1, from germinating tomato seeds that
exhibits high predicted amino acid sequence homology to NPK5 of tobacco and
StubSNF1 of potato, as would be expected from their close taxonomic
relationship. In addition, LeSNF1 complemented an snf1
mutant of yeast, restoring both growth on Suc and derepression of invertase
(Figs. 4B and
5B) and indicating that it
encodes a functional kinase that can respond to Glc repression signals in
yeast. Strong binding of LeSNF1 to yeast SNF4 in the two-hybrid assay only
under low-Glc conditions further supports this conclusion
(Table I). Like yeast SNF1
(Celenza and Carlson, 1989)
and Arabidopsis AKIN10 and AKIN11 (Bhalerao
et al., 1999), but unlike tobacco NPK5
(Muranaka et al., 1994),
LeSNF1 also suppressed an snf4 mutation in yeast (Figs.
4A and
5A). Suppression of
snf4 is likely due to overexpression of LeSNF1 or SNF1 kinase
activity, resulting in induction of some invertase activity even in the
presence of high Glc concentrations (Fig.
5). However, invertase expression was stimulated severalfold under
low-Glc conditions in yeast snf4 mutants expressing LeSNF1
(Fig. 5A), indicating that like
SNF1 (Jiang and Carlson,
1996), LeSNF1 remains responsive to signals regulating the Glc
repression system in yeast even in the absence of SNF4. Interestingly,
interaction of LeSNF1 protein with itself in the two-hybrid assay was enhanced
under low-Glc conditions (Table
I). In contrast, internal binding of the SNF1 regulatory and
kinase domains in yeast is enhanced in high Glc
(Jiang and Carlson, 1996).
Apparent interaction of LeSNF1 with itself also could be due to the presence
of endogenous yeast proteins that could bridge between two LeSNF1 proteins in
the two-hybrid system under low-Glc conditions.
In contrast to the many known plant SnRK1- homologs
(Halford et al., 2000),
relatively few candidate genes for the - and -subunits of the
complex have been identified. The first plant -subunit candidate,
termed Pv42, was isolated serendipitously from bean
(Abe et al., 1995), and no
further information on this gene has been reported. Bouly et al.
(1999) used sequence homology
with SNF4 and AMPK to isolate an Arabidopsis gene
(AKIN) whose protein product interacted with AKIN1 in
two-hybrid assays. However, the sequence of AKIN is quite divergent
from other plant -subunit candidates
(Fig. 1B), and it did not
interact with yeast SNF1 protein or complement a yeast snf4 mutant
(Bouly et al., 1999), so
whether it functions in vivo as part of an SnRK1 complex is uncertain.
Similarly, Slocombe et al.
(2002) identified an
SnRK1-interacting protein (SnIP1) having slight similarity to other plant
SnRK1- candidates that also was unable to complement a yeast
snf4 mutant. Another candidate -subunit gene (AtSNF4)
was identified from Arabidopsis as a "weak" suppressor of the
yeast snf4 mutation (Kleinow et
al., 2000). It was subsequently shown that expression of a longer
transcript of this gene containing a 5' KIS domain in addition to the
-subunit domain effectively complemented the yeast snf4
mutation (Lumbreras et al.,
2001). Similarly, both the full-length ZmAKIN-1
protein from maize and the -related region alone complemented the yeast
snf4 mutation and interacted with AKIN11, an SnRK1- protein
(Lumbreras et al., 2001).
Because the only functional plant homologs of SNF4 known at that time were of
the -type, these authors proposed that plant SnRK1 complexes may
consist of dimers of - and fused -type subunits
(Lumbreras et al., 2001).
We identified a putative SNF4 homolog from tomato (LeSNF4) that
exhibited sequence homology to both SNF4 and AMPK- and to other
potential plant SnRK1- proteins
(Fig. 1). The tomato protein
complemented a yeast snf4 deletion mutant and restored derepression
of invertase during Glc starvation (Figs.
4A and
5A). The expressed protein
interacted strongly with both yeast SNF1 and LeSNF1 proteins only under
low-Glc conditions (Table I).
The LeSNF4 gene encodes only a -type subunit and does not
contain -related sequences in the 1,234-bp 5' to the translation
start site or in the expressed mRNA. We also identified two tomato cDNAs
(LeSIP1 and LeGAL83) containing conserved KIS and ASC
domains characteristic of -subunits
(Fig. 3), and LeSIP1
exhibited Glc-sensitive binding to LeSNF4 and LeSNF1 and to itself
(Table I). We are not aware of
any other reports testing whether -subunits can self-associate under
low-Glc conditions. We have not tested LeGAL83 in the two-hybrid
assay and were unable to achieve meaningful results with StubGAL83 in
that assay, but StubGAL3 was previously reported to interact with StubSNF1 and
SNF4 (Lakatos et al., 1999).
Thus, although plants possess genes for chimeric -type proteins
that can interact with SnRK1- proteins
(Lumbreras et al., 2001), they
also contain genes encoding separate functional - and -type
subunits analogous to those in yeast and mammals.
Halford et al. (2000,
2003) have discussed some of
the potential biochemical differences between the plant SnRK1 complexes and
those of yeast and mammals, such as the likelihood that Suc, rather than Glc,
may be the primary sugar regulating the activity of the kinase in plants.
Plant SnRK1 kinases are involved in multiple signal transduction and/or
regulation pathways, and a number of proteins in addition to SnRK1- and
- subunits have been identified that could potentially interact with
SnRK1 kinases to influence their activity or substrate specificity (e.g.
Bhalerao et al., 1999;
Farrás et al., 2001;
Fordham-Skelton et al., 2002;
Slocombe et al., 2002).
Biochemical studies are needed to identify the in vivo substrates for LeSNF1
and whether its activity, localization, and/or substrate specificity are
altered by the presence of LeSNF4, SIPs, or sugars. However, the high sequence
conservation among eukaryotic SnRK1 components, the ability of LeSNF1 and
LeSNF4 to interact with each other and with their yeast counterparts, and
their ability to respond to Glc derepression signals in yeast all suggest that
these proteins are likely to be involved in regulating carbon metabolism in
planta. We are developing transgenic tomato plants in which LeSNF4 expression
can be experimentally modified to test this possibility.
LeSNF4 Is Differentially Expressed during Seed Development
and Germination and in Response to Hormones and Water Stress
Relatively limited information is available about the expression of genes
of the SnRK1 complex in plants. Different SnRK1--subunit genes are
expressed in vegetative and seed tissues of barley (Hordeum vulgare),
and a specific subfamily (SnRK1b) is uniquely expressed in cereal seeds
(Hannappel et al., 1995;
Takano et al., 1998;
Halford et al., 2000). Potato
PKIN1 showed somewhat higher expression in stolons than in other
tissues (Man et al., 1997). In
Arabidopsis, AKIN1, AKIN,
AKIN1, and AKIN2 all exhibited
low expression in siliques, whereas mRNA abundance of
AKIN1 in leaves may be regulated by light
(Bouly et al., 1999).
Abundance of StubSNF1 mRNA was low in various plant tissues except
flowers, where expression was high, whereas StubGAL83 mRNA was
detected in all tissues tested, with lower expression in roots and fruits
(Lakatos et al., 1999).
AtSNF4 mRNA was most abundant in cell suspension cultures, flower
buds, and shoot apices (Kleinow et al.,
2000). ZmAKIN-1 mRNA was most
abundant during early embryogenesis, and its expression in seedlings was not
affected by ABA or temperature stress (4°C or 37°C;
Lumbreras et al., 2001).
Tobacco NPK5 and potato PKIN1 exhibited some variation in
mRNA abundance among tissues, but their mRNAs were detected in all tissues
tested (Muranaka et al., 1994;
Man et al., 1997). Thus,
studies of the expression of genes of the plant SnRK1 complex have been
limited largely to tissue surveys and some alterations of environmental
conditions, and in only one study were putative members of all three SnRK1
components examined (Bouly et al.,
1999).
The dramatic changes in carbon fluxes associated with the deposition of
reserves during seed development and their mobilization during germination
provide an opportunity to test whether expression of the components of the
SnRK1 complex are altered during these metabolic transitions. During tomato
seed development, LeSNF4 mRNA was initially absent but appeared at 30
d after flowering, which coincided with maximum dry weight accumulation and
the first appearance of planteose, a Suc-galactosyl oligosaccharide that
accumulates late in tomato seed development
(Fig. 7;
Downie et al., 2003). The
increase in LeSNF4 mRNA coincided with increasing ABA concentrations
in tomato seeds between 30 and 40 DAF, and both ABA content and
LeSNF4 mRNA remained high thereafter
(Fig. 7, B and C;
Hocher et al., 1991;
Berry and Bewley, 1992).
LeSNF4 mRNA was present in imbibed seeds under various conditions
where germination was inhibited and was absent from seeds after completion of
germination (Figs. 6 and
8). In contrast,
LeSNF1 mRNA was present throughout seed development, being the most
abundant in mature seeds (Fig. 7, B and
C), and LeSNF1 mRNA amounts were essentially constant
after imbibition regardless of germination status
(Fig. 8). Comparable results
were found with true (botanical) potato seeds when LeSNF4 and
LeSNF1 probes were used to detect homologous potato mRNAs in similar
types of experiments (Alvarado et al.,
2000). Although represented in an imbibed seed cDNA library and in
a tomato ovary EST library, suggesting that it is not a pseudogene, we were
unable to detect LeSIP1 mRNA in developing or germinating seeds or
unstressed leaves by hybridization, whereas LeGAL83 mRNA was detected
during late seed development and throughout germination (Figs.
7,
8,
9). Different -subunits
of the SnRK1 complex apparently are differentially expressed in a
tissue-specific manner, as shown previously for human and Arabidopsis genes
(Thornton et al., 1998;
Bouly et al., 1999).
LeGAL83 mRNA abundance was low during early seed development but
increased as seeds approached maturity
(Fig. 7, B and C), and like
LeSNF1, remained relatively constant in imbibed seeds, regardless of
the germination status (Fig.
8). We conclude that at least one SnRK1- gene
(LeSNF1) and one SnRK1- gene (LeGAL83) are expressed
in developing and germinating tomato seeds and that their mRNA abundance is
relatively unaffected by conditions that markedly alter expression of
LeSNF4.
The expression pattern of LeSNF4 is reminiscent of that of an
SnRK2 kinase, PKABA1, which was identified from wheat embryos on the
basis of its induction by ABA (Anderberg
and Walker-Simmons, 1992). PKABA1 is involved in a signaling
pathway by which ABA suppresses the induction of gene expression by GA in
cereal aleurone layers (Gomez-Cadenas et
al., 2001). Like LeSNF4, PKABA1 mRNA remains abundant in
imbibed dormant seeds, but declines soon after imbibition in nondormant seeds
(Verhey and Walker-Simmons,
1997). Furthermore, its expression is up-regulated by ABA,
dehydration, cold, and osmotic stress in other plant tissues
(Holappa and Walker-Simmons,
1995). Similarly, we found that expression of the LeSNF4
gene can be stimulated in leaves by ABA or water stress
(Fig. 9). This and the decrease
in LeSNF4 mRNA after GA treatment of gib-1 seeds
(Fig. 6) are consistent with
direct regulation of LeSNF4 expression by these hormones and with the
presence of consensus ABA- and GA-responsive elements in the promoter region
of the LeSNF4 gene.
Regulation of LeSNF4 Expression Could Link Hormonal and
Sugar Response Pathways
Our discovery that ABA, GA, and other conditions that influence seed
germination regulate LeSNF4 expression provides a possible mechanism
by which hormonal and sugar-signaling pathways could interact. In yeast and
mammals, the SNF1/AMPK kinase complex functions as a metabolic switch, acting
on both key enzymes and transcription factors to either induce or repress
entire metabolic pathways in response to nutritional or environmental stress
or energy demand (Carlson,
1998; Hardie et al.,
1998). Rook et al.
(2001) proposed that ABA
signaling modulates responses to a separate sugar signal, suggesting an
explanation for the altered sensitivity of germination to inhibition by sugars
in several aba and abi mutants
(Cheng et al., 2002;
Finkelstein et al., 2002;
Rolland et al., 2002). Rook et
al. (2001) also proposed that
interactions between ABA and sugar-signaling pathways shift development and
metabolism between a "storage mode" in which storage reserves are
synthesized and their utilization is inhibited and a "mobilization
mode" in which storage reserves are catabolized and growth is promoted.
Similarly, Gazzarrini and McCourt
(2001) proposed that
progression from germination to seedling growth involves a transition from
"State 1" in which ABA or sugars are capable of blocking reserve
mobilization and germination to "State 2" or autotrophic
vegetative growth that is much less sensitive to ABA or sugars. The well-known
antagonism between ABA and GA in cereal aleurone cells
(Gomez-Cadenas et al., 2001)
can also be viewed as switching metabolism between the "storage"
and "mobilization" modes. Consistent with its role in other
organisms and its expression pattern during seed development and germination,
we propose that modulation of LeSNF1 kinase activity by the ABA-regulated
expression of LeSNF4 could be involved in this metabolic shift
(Fig. 10).
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Figure 10. Regulation and hypothetical roles of the SnRK1 complex during seed
maturation and germination. This scheme combines recent models (e.g.
Gazzarrini and McCourt, 2001;
Rook et al., 2001) with data
presented here for the tomato SnRK1 complex. ABA synthesis during seed
development or in response to high sugar levels during germination results in
ABA accumulation, inducing LeSNF4 expression. Additional factors promoting
LeSNF4 expression are shown on the left of the ABA signaling pathway, whereas
those inhibiting expression are shown on the right. Dashed lines both above
and below LeSNF4 are shown because it is unclear where each of these factors
acts in influencing expression. When LeSNF4 is present, it binds with
LeSNF1/LeGAL83, potentially altering the kinase activity or its interaction
with other regulatory factors or substrates, resulting in the maintenance of a
"maturation/dormancy" metabolic state and inhibiting reserve
mobilization. When LeSNF4 is absent, LeSNF1 may have altered activity,
specificity, or interactions with other SIPs, resulting in transition to the
"germination/growth" mode required for reserve mobilization and
seedling growth. In either of the metabolic modes, the activity of the LeSNF1
complex may be sensitive to regulation by sugars (indicated by the broken
lines) and could be involved in the regulation of ABI3, ABI4, and ABI5 and
other proteins that are known to influence the transition from seed maturation
and dormancy to germination and growth. (Arrows indicate promotion; bars
indicate inhibition.)
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We hypothesize that when LeSNF4 is expressed during seed maturation
(probably in response to ABA), it would bind to LeSNF1/LeGAL83 (or other SIP
proteins) and alter the kinase activity of the complex to promote metabolic
pathways involved in the accumulation or maintenance of storage reserves and
to block those involved in the mobilization or utilization of stored reserves
(Fig. 10). For example, LeSNF4
might modulate LeSNF1 activity or substrate specificity to express metabolic
pathways that result in the reduction in Glc and accumulation of Suc and
oligosaccharides such as raffinose and planteose that occur during seed
maturation. After imbibition, expression of LeSNF4 is reduced in seeds that
are not dormant or are stimulated by GA, potentially altering LeSNF1 kinase
activity to derepress genes encoding enzymes required for reserve mobilization
and metabolism (Fig. 10).
Conditions that block germination of imbibed seeds, including dormancy, ABA,
FR light, or low water potential, maintain LeSNF4 expression
(Fig. 10). Provision of sugars
can overcome the initial inhibition of germination by ABA
(Garciarrubio et al., 1997;
Finkelstein and Lynch, 2000a)
and induce germination in embryos of the cts (comatose)
mutant, which are deeply dormant due to a defect in lipid reserve mobilization
(Footitt et al., 2002).
Interestingly, ABA did not entirely block lipid mobilization in imbibed
Arabidopsis seeds but decreased utilization of Suc, resulting in a doubling of
Suc content (Pritchard et al.,
2002). ABA action on supply or utilization of metabolic substrates
is also consistent with the lack of effect of ABA on the induction of
germination-associated cell wall hydrolases and expansins by GA or on the
initial weakening of the endosperm cap enclosing the radicle tip of tomato
seeds, nonetheless preventing germination
(Bradford et al., 2000;
Chen and Bradford, 2000).
The developmental arrest associated with embryo quiescence and dormancy
that separates the maturation and germination modes requires ABA and is absent
in mutants of genes such as ABI3, FUS3, LEC1, and LEC2,
which encode transcription factors that influence the expression of a number
of genes associated with seed maturation and germination
(Nambara et al., 2000;
Raz et al., 2001;
Koornneef et al., 2002).
Additional transcription factors that can bind ABA-responsive elements (ABREs)
have been identified in plants, including ABI5, ABRE-binding factors (ABFs),
and ABRE-binding proteins (AREBs; Choi et
al., 2000; Uno et al.,
2000; Carles et al.,
2002). In Arabidopsis, ABI5 acts specifically at the
postgermination transition in an ABA-dependent manner to maintain the embryo
in a quiescent state and block further seedling development
(Finkelstein and Lynch, 2000b;
Lopez-Molina et al., 2001,
2002). Overexpression of ABF3
exerted an inhibitory effect on germination and early seedling growth and
conferred hypersensitivity to ABA (Kang et
al., 2002), similar to the effects of ectopic ABI4 expression
(Söderman et al., 2000).
Genetic and molecular data indicate that ABI3-, ABI4-, and ABI5-related
proteins are involved in an interactive network regulating late seed
development and germination (Söderman
et al., 2000; Nakamura et al.,
2001; Bensmihen et al.,
2002; Brocard et al.,
2002; Niu et al.,
2002). Many of these genes are transcriptionally and/or
posttranslationally regulated by ABA (e.g.
Lopez-Molina et al., 2003),
and some of them, including ABI5 (also known as TRAB1), ABF2 (also known as
AREB1), and ABF4 (also known as AREB2), are phosphorylated in response to ABA
(Uno et al., 2000;
Lopez-Molina et al., 2001,
2002;
Kagaya et al., 2002;
Lu et al., 2002). No plant
transcription factors have been demonstrated to be substrates for SnRK1 kinase
activity, but the two Ser (S42 and S145) and one Tyr (T201) that are
phosphorylated in ABI5 (Lopez-Molina et
al., 2002) and Ser-102 in TRAB1 that is phosphorylated in response
to ABA and is essential for ABA-induced transcription
(Kagaya et al., 2002) are all
found in consensus recognition sequences for phosphorylation by SnRK1
(Halford et al., 2003).
Interestingly, the same consensus phosphorylation recognition sequence is
present in a region of the Mig1 transcriptional repressor in yeast that is
required for its regulation by SNF1 in the absence of Glc
(Östling et al., 1996).
In addition, a wheat transcription factor, TaABF, related to the ABI5 family,
was recently identified as a substrate for PKABA1
(Johnson et al., 2002).
Although experimental data are as yet lacking, an intriguing possibility is
that LeSNF1 is involved in the kinase cascades that phosphorylate the
transcription factors regulating late embryogenesis, dormancy, and the
transition to germination. Two-hybrid interaction assays with ABI3- and
ABI5-related proteins (e.g. Nakamura et
al., 2001) conducted in yeast both with and without Glc could be
informative in this regard. ABA and/or sugars, acting directly or via
expression of LeSNF4, could alter the activity, substrate specificity, or
localization of the SnRK1 complex to phosphorylate alternative transcription
factors resulting in either the maturation/dormancy or germination/growth
metabolic states (Fig.
10).
In conclusion, our results indicate that mRNA abundance of the
-subunit of the SnRK1 complex in tomato (LeSNF4) is regulated by ABA
and GA in a manner consistent with its having a functional role in the
maturation to growth transition in seeds and in stress responses in leaves. By
adding hormonal regulation to the sugar-, energy-, and stress-sensing
functions of a prototypical SnRK1 complex, plants apparently have adapted it
for a variety of functions related to source/sink relationships, developmental
transitions, and environmental responses.
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MATERIALS AND METHODS
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Isolation of LeSNF4, LeSNF1, and LeSIP1
Differential cDNA display was performed essentially as described by Liang
and Pardee (1992), using mRNA
pools from GA-deficient (gib-1) tomato (Lycopersicon
esculentum Mill.) seeds (Groot and
Karssen, 1987) imbibed in water or in 100 µM
GA4+7 for 24 h
(Cooley et al., 1999). Anchor
primer (dT15MG, where M = G, A, or C) and an arbitrary primer
(5'AATCGGGCTG3') amplified a 300-bp cDNA fragment that was present
only in seeds imbibed on water. This fragment was used to retrieve a partial
cDNA from a library prepared using mRNA from gib-1 seeds incubated
for 24 h on water (Zap Express cDNA Synthesis Kit, Stratagene, La Jolla, CA).
The full-length cDNA was obtained using 5'-RACE (Life Technologies,
Gaithersburg, MD). The complete cDNA was subsequently used to isolate a single
3.5-kb fragment from a tomato cv VFNT Cherry genomic library in Charon 35
vector (courtesy of R.L. Fischer, University of California, Berkeley). This
genomic DNA contained the complete LeSNF4 coding region, including
two introns, and 1,234 bp in the promoter region.
LeSNF1
cDNA was isolated from a library prepared using mRNA from gib-1
seeds incubated for 24 h on 100 µM
GA4+7 (Zap Express cDNA Synthesis Kit,
Stratagene) using tobacco (Nicotiana tabacum) NPK5 cDNA as a
probe (courtesy of Dr. Toshiya Muranaka, Sumitomo Chemical Co., Ltd.,
Takarazuka, Hyogo 665, Japan). LeSIP1 cDNA was isolated from the same
library using tomato EST AI486580 (provided by Clemson University Genomics
Institute, SC) as a probe. The partial cDNA of LeGAL83 was isolated
by reverse transcription-PCR using primers based on the StubGAL83
sequence. cDNAs were sequenced at the Advanced Plant Genetics Facility
(University of California, Davis). Sequence comparisons were made using
DNAStar software.
Yeast (Saccharomyces cerevisiae) Complementation
LeSNF4 or LeSNF1 cDNAs were fused in frame with pMA424
and pYES and these or empty vectors were transformed into freshly prepared
snf4-2 (MCY3915 [MAT snf4-2,
His-3200 Leu-2-3, and 112-Trp 163 Ura-3-52]) or
snf1-10 (MCY1846 [MAT
snf1-10 Lys-2-801 Ura 3-52]) yeast cells (provided by
Dr. Marian Carlson, Columbia University, New York) or wild-type YPH500
competent cells by the lithium acetate method
(Rose et al., 1990). The
transformed yeast colonies were plated on selective SC medium at 29°C for
3 to 14 d, and growing colonies were characterized by invertase assays and by
re-isolation and restriction analysis of the shuttle vector to confirm the
presence of the inserted genes.
Invertase Assays
Yeast cells were grown in 3 mL of selective medium at 29°C and
harvested at mid-log phase. After centrifuging for 5 min at 15,000g
and noting the weight of the pellet, the cells were chilled in ice and washed
with 100 µL of chilled sterile water. The cells were resuspended in 100
µL of glass bead disruption buffer and shaken 6 x 1 min each with 80
mg of cooled glass beads (Sigma, St. Louis). The supernatant was spun at
12,000g for 30 min to yield the crude extract. Extract (4 µL) was
assayed according to Goldstein and Lampen
(1975; except that
O-dianisidine was dissolved in methanol) using invertase (Sigma) as
the standard.
Yeast Two-Hybrid Protein Interaction Assays
In-frame fusions of members of the SnRK1 complex from tomato and yeast were
made by PCR in pGBT9.BS (Trp; binding domain) and pGAD.GH (Leu; activation
domain) vectors. Competent Y190 cells were transformed as above with equal
quantities (0.25–0.5 µg) of the two vectors. The cells were plated on
SC-Trp-Leu-His with 25 mM 3-amino-triazole and grown at 29°C
for 3 to 14 d. The transformed colonies were characterized by filter lift
assays for -galactosidase, and colonies showing blue color were grown in
3 mL of selective broth containing 2% (w/v) Glc or 2% (w/v) each of Gal,
glycerol, and ethanol plus 0.05% (w/v) Glc with 25 mM
3-aminotriazole. -D-Galactosidase was assayed
spectrophotometrically in extracts (as described above for invertase) using
chlorophenyl-red--D-galactopyranoside as substrate
(Kim et al., 1997).
Galactosidase activity was calculated using an extinction coefficient for
chlorophenyl red at 570 nm of 75,000 L
mol–1cm–1.
Seed Development
Tomato cv Moneymaker plants were grown in the field in the summer of 2000.
Flowers were marked at anthesis, and fruits were harvested every 5 d between
20 and 60 DAF. Seeds were separated from the locular tissue by gentle rubbing,
and RNA was extracted without drying the seeds.
Northern and Southern Hybridization
Northern hybridizations were conducted as described previously using
digoxigenin-labeled RNA probes (Cooley et
al., 1999). Hybridization was detected according to the
instructions in the Genius System
(Boehringer Mannheim Corporation,
1995). All experiments involving mRNA abundance were repeated at
least twice with similar results. Southern hybridizations with DNA probes were
conducted as described previously (Chen
and Bradford, 2000) and detected using enhanced chemiluminescence
(ECL kit, Amersham Life Science, Arlington Heights, IL). After hybridization,
membranes were washed twice at 42°C with 6 M urea, 0.5% (w/v)
SDS, and then washed twice for 5 min each with 2x SSC at room
temperature.
|
ACKNOWLEDGMENTS
|
|---|
We thank Dr. Marian Carlson for providing the mutant yeast strains, Dr.
Linda Bisson for wild-type yeast YPH500, Dr. Toshiya Muranaka for providing
the cDNA of NPK5, Dr. Zsófia Bánfalvi for providing the
cDNA of StubGAL83, and the Clemson University Genomics Institute for
providing the cDNA of EST AI486580. Dr. Steffen Abel provided valuable advice
on the yeast complementation and two-hybrid assays.
GA4+7 was a gift from Abbott Laboratories
(Chicago).
Received December 15, 2002;
returned for revision January 20, 2003;
accepted March 12, 2003.
|
FOOTNOTES
|
|---|
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.102.019141.
1 This work was supported in part by the National Science Foundation (grant
nos. IBN–9407264 and IBN–9722978) and by the U.S. Department of
Agriculture-National Research Initiative Competitive Grants Program (grant no.
2001–35304 to K.J.B.). 
3 Present address: Department of Horticulture, N322–C Agricultural
Science Center North, University of Kentucky, Lexington, KY
40546–0091.
2 Deceased.
4 Present address: Department of Biology, BSBE 201, Texas A&M University,
College Station, TX 77843–3258.
5 Present address: 945 Lucena Court, Davis, CA 95616.
*
Corresponding author; e-mail
kjbradford{at}ucdavis.edu;
fax 530–752–4554.
|
LITERATURE CITED
|
|---|
Abe H, Kamiya Y, Sakurai A (1995) A cDNA clone
encoding yeast SNF4-like protein (Accession #U40713) from Phaseolus
vulgaris L. Plant Physiol
110: 336
Alderson A, Sabelli PA, Dickinson JR, Cole D, Richardson M,
Kreiss M, Shewry PR, Halford NG (1991) Complementation
of snf1, a mutation affecting global regulation of carbon metabolism in yeast,
by a plant protein kinase cDNA. Proc Natl Acad Sci USA
88:
8602–8605[Abstract/Free Full Text]
Alvarado V, Nonogaki H, Bradford KJ (2000)
Expression of endo--mannanase and SNF-related protein kinase genes in
true potato seeds in relation to dormancy, gibberellin and abscisic acid.
In JD Viemont, J Crabbé, eds, Dormancy in
Plants. CAB International, Wallingford, UK, pp
347–364
Anderberg RJ, Walker-Simmons MK (1992)
Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with
homology to protein kinases. Proc Natl Acad Sci USA
89:
10183–10187[Abstract/Free Full Text]
Bateman A (1997) The structure of a domain
common to archaebacteria and the homocystinuria disease protein. Trends
Biochem Sci 22:
12–13[Web of Science][Medline]
Bensmihen S, Rippa S, Lambert G, Jublot D, Pautot V, Branier F,
Giraudat J, Parcy F (2002) The homologous ABI5 and EEL
transcription factors function antagonistically to fine-tune gene expression
during late embryogenesis. Plant Cell
14:
1391–1403[Abstract/Free Full Text]
Berry T, Bewley JD (1991) Seeds of tomato
(Lycopersicon esculentum Mill.) which develop in a fully hydrated
environment in the fruit switch from the developmental to germination mode
without a requirement for desiccation. Planta
186:
27–34
Berry T, Bewley JD (1992) A role for the
surrounding fruit tissues in preventing germination of (Lycopersicon
esculentum) seeds: a consideration of osmotic environment and abscisic
acid. Plant Physiol 100:
951–957[Abstract/Free Full Text]
Bhalerao RP, Salchert K, Bakó L, Ökrész L,
Szabados L, Muranaka T, Machida Y, Schell J, Koncz C
(1999) Regulatory interaction of PRL1 WD protein with Arabidopsis
SNF1-like protein kinases. Proc Natl Acad Sci USA
96:
5322–5327[Abstract/Free Full Text]
Boehringer Mannheim Corporation (1995)
Genius System User's Guide for Membrane Hybridization, Version
3.0. Boehringer Mannheim, Indianapolis
Bouly J, Gissot L, Lessard P, Kreis M, Thomas M
(1999) Arabidopsis thaliana proteins related to the
yeast SIP and SNF4 interact with AKIN1, an SNF1-like protein kinase.
Plant J 18:
541–550[CrossRef][Web of Science][Medline]
Bradford KJ, Chen F, Cooley MB, Dahal P, Downie B, Fukunaga KK,
Gee OH, Gurusinghe S, Mella RA, Wu CT et al. (2000)
Gene expression prior to radicle emergence in imbibed tomato seeds.
In M Black, KJ Bradford, J Vázquez-Ramos, eds, Seed
Biology: Advances and Applications. CABI Publishing, Wallingford, UK,
pp 231–251
Brocard IM, Lynch TJ, Finkelstein RR (2002)
Regulation and role of the Arabidopsis abscisic acid-insensitive5
gene in abscisic acid, sugar, and stress response. Plant
Physiol 129:
1533–1543[Abstract/Free Full Text]
Carles C, Bies-Etheve N, Aspart L, Léon-Kloosterziel KM,
Koornneef M, Echeverria M, Delseny M (2002) Regulation
of Arabidopsis thaliana Em genes: role of ABI5. Plant
J 30:
373–383[CrossRef][Web of Science][Medline]
Carlson M (1998) Regulation of Glc utilization
in yeast. Curr Opin Genet Dev 8:
560–564[CrossRef][Web of Science][Medline]
Celenza JL, Carlson M (1989) Mutational
analysis of the Saccharomyces cerevisiae SNF1 protein kinase and
evidence for functional interaction with the SNF4 protein. Mol Cell
Biol 9:
5034–5044[Abstract/Free Full Text]
Celenza JL, Eng FJ, Carlson M (1989) Molecular
analysis of the SNF4 gene of Saccharomyces cerevisae: evidence for
physical association of SNF4 protein with the SNF1 protein kinase. Mol
Cell Biol 9:
5045–5054[Abstract/Free Full Text]
Chen F, Bradford KJ (2000) Expression of an
expansin is associated with endosperm weakening during tomato seed
germination. Plant Physiol 124:
1265–1274[Abstract/Free Full Text]
Chen FQ, Foolad MR (1999) A molecular linkage
map of tomato based on a cross between Lycopersicon esculentum and
L. pimpinellifolium and its comparison with other
molecular maps of tomato. Genome 42:
94–103
Cheng W-H, Endo A, Zhou L, Penney J, Chen H-C, Arroyo A, Leon
P, Nambara E, Asami T, Seo M et al. (2002) A unique
short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and
abscisic acid biosynthesis and functions. Plant Cell
14:
2723–2743[Abstract/Free Full Text]
Choi HI, Hong JH, Ha JO, Kang JY, Kim SY (2000)
ABFs, a family of ABA-responsive element binding factors. J Biol
Chem 275:
1723–1730[Abstract/Free Full Text]
Cooley MB, Yang H, Dahal P, Mella RA, Downie B, Haigh AM,
Bradford KJ (1999) Expression of vacuolar
H+-ATPase in response to gibberellin during tomato seed
germination. Plant Physiol 121:
1339–1347[Abstract/Free Full Text]
Crawford RM, Halford NG, Hardie DG (2001)
Cloning of DNA encoding a catalytic subunit of SNF1-related protein kinase-1
(SnRK1-1), and immunological analysis of multiple forms of the kinase,
in spinach leaf. Plant Mol Biol
45:
731–741[CrossRef][Web of Science][Medline]
Debeaujon I, Koornneef M (2000) Gibberellin
requirement for Arabidopsis seed germination is determined both by testa
characteristics and embryonic abscisic acid. Plant Physiol
122:
415–424[Abstract/Free Full Text]
Downie B, Gurusinghe S, Dahal P, Thacker RR, Snyder JC, Nonogaki
H, Yim K, Fukanaga K, Alvarado V, Bradford KJ (2003)
Expression of a galactinol synthase gene in tomato is stress induced by an
ABA-independent mechanism that is differentially responsive to cold in tomato
seeds and leaves. Plant Physiol
131:
1347–1359[Abstract/Free Full Text]
Farrás R, Ferrando A, Jásik J, Kleinow T,
Ökréaz L, Tiburcio A, Salchert K, del Pozo C, Schell J, Koncz
C (2001) SKP1-SnRK protein kinase interactions mediate
proteasomal binding of a plant SCF ubiquitin ligase. EMBO J
20:
2742–2756[CrossRef][Web of Science][Medline]
Ferrando A, Koncz-Kálmán Z, Farràs R,
Tiburcio A, Schell J, Koncz C (2001) Detection of in
vivo protein interactions between Snf1-related kinase subunits with
intron-tagged epitope-labelling in plants cells. Nucleic Acids
Res 29:
3685–3693[Abstract/Free Full Text]
Finkelstein RR, Gampala SSL, Rock CD (2002)
Abscisic acid signaling in seeds and seedlings. Plant Cell
14:
S15–S45[Free Full Text]
Finkelstein RR, Gibson SI (2001) ABA and sugar
interactions regulating development: cross-talk or voices in a crowd?
Curr Opin Plant Biol 5:
26–32
Finkelstein RR, Lynch TJ (2000a) Abscisic acid
inhibition of radicle emergence but not seedling growth is suppressed by
sugars. Plant Physiol 122:
1179–1186[Abstract/Free Full Text]
Finkelstein RR, Lynch TJ (2000b) The
Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine
zipper transcription factor. Plant Cell
12:
599–609[Abstract/Free Full Text]
Footitt S, Slocombe SP, Larner V, Kurup S, Wu Y, Larson T,
Graham I, Baker A, Holdsworth M (2002) Control of
germination and lipid mobilization by COMATOSE, the
Arabidopsis homologue of human ALDP. EMBO J
21:
2912–2922[CrossRef][Web of Science][Medline]
Fordham-Skelton AP, Chilley P, Lumbreras V, Reignoux S, Fenton
TR, Dahm CC, Pages M, Gatehouse JA (2002) A novel
higher plant protein tyrosine phosphatase interacts with SNF1-related protein
kinases via a KIS (kinase interaction sequence) domain. Plant J
29:
705–715[CrossRef][Web of Science][Medline]
Gallardo K, Job C, Groot SPC, Puype M, Demol H, Vandekerckhove
J, Job D (2002) Proteomics of Arabidopsis seed
germination: a comparative study of wild-type and gibberellin-deficient seeds.
Plant Physiol 129:
823–837[Abstract/Free Full Text]
Garciarrubio A, Legaria JP, Covarrubias AA
(1997) Abscisic acid inhibits germination of mature
Arabidopsis seeds by limiting the availability of energy and
nutrients. Planta 203:
182–187[CrossRef][Web of Science][Medline]
Gazzarrini S, McCourt P (2001) Genetic
interactions between ABA, ethylene and sugar signaling pathways. Curr
Opin Plant Biol 4:
387–391[CrossRef][Web of Science][Medline]
Gibson SI (2000) Plant sugar-response pathways:
part of a complex regulatory web. Plant Physiol
124:
1532–1539[Free Full Text]
Girke T, Todd J, Ruuska S, White J, Benning C, Ohlrogge J
(2000) Microarray analysis of developing Arabidopsis seeds.
Plant Physiol 124:
1570–1581[Abstract/Free Full Text]
Goldstein A, Lampen JO (1975)
-D-Fructofuranoside fructohydrolase from yeast.
Methods Enzymol 42C:
504–511[Medline]
Gomez-Cadenas A, Zentella R, Walker-Simmons MK, Ho THD
(2001) Gibberellin/abscisic acid antagonism in barley aleurone
cells: site of action of the protein kinase PKABA1 in relation to gibberellin
signaling molecules. Plant Cell
13:
667–679[Abstract/Free Full Text]
Groot SPC, Karssen CM (1987) Gibberellins
regulate seed germination in tomato by endosperm weakening: a study with
gibberellin-deficient mutants. Planta
171:
525–531[CrossRef][Web of Science]
Halford NG, Bouly J-P, Thomas M (2000)
SNF1-related protein kinases (SnRKs): regulators at the heart of the control
of carbon metabolism and partitioning. Adv Bot Res
32:
405–434
Halford NG, Hardie DG (1998) SNF1-related
protein kinases: global regulators of carbon catabolism in plants?
Plant Mol Biol 37:
735–748[CrossRef][Web of Science][Medline]
Halford NG, Hey S, Jhurreea D, Laurie S, McKibbin RS, Paul M,
Zhang Y (2003) Metabolic signalling and carbon partitioning:
role for Snf1-related (SnRK1) protein kinase. J Exp Bot
54:
467–475[Abstract/Free Full Text]
Hannappel U, Vicente-Carbajosa J, Barker JHA, Shewry PR, Halford
NG (1995) Differential expression of two barley SNF1-related
protein kinase genes. Plant Mol Biol
27:
1235–1240[CrossRef][Web of Science][Medline]
Hardie DG, Carling D, Carlson M (1998) The
AMP-activated/SNF1-protein kinase subfamily: metabolic sensors of the
eukaryotic cell? Annu Rev Biochem
67:
821–855[CrossRef][Web of Science][Medline]
Hocher V, Sotta B, Maldiney R, Miginiac E
(1991) Changes in abscisic acid and its
-D-glucopyranosyl ester levels during tomato
(Lycopersicon esculentum Mill.) seed development. Plant Cell
Rep 10:
444–447
Holappa LD, Walker-Simmons MK (1995) The wheat
abscisic acid-responsive protein kinase mRNA, PKABA1, is up-regulated by
dehydration, cold temperature, and osmotic stress. Plant
Physiol 108:
1203–1210[Abstract]
Jiang R, Carlson M (1996) Glucose regulates
protein interactions within the yeast SNF1 protein kinase complex.
Genes Dev 10:
3105–3115[Abstract/Free Full Text]
Jiang R, Carlson M (1997) The Snf1 protein
kinase and its activating subunit, Snf4, interact with distinct domains of the
Sip1/Sip2/Gal83 component in the kinase complex. Mol Cell Biol
17:
2099–2106[Abstract/Free Full Text]
Johnson R, Wagner R, Verhey SD, Walker-Simmons MK
(2002) The abscisic acid-responsive kinase PKABA1 interacts with
a seed-specific ABA response element-binding factor, TaABF, and phosphorylates
TaABF peptide sequences. Plant Physiol
130:
837–846[Abstract/Free Full Text]
Kagaya Y, Hobo T, Murata M, Ban A, Hattori T
(2002) Abscisic acid-induced transcription is mediated by
phosphorylation of an abscisic acid response element binding factor, TRAB1.
Plant Cell 14:
3177–3189[Abstract/Free Full Text]
Kang JY, Choi HI, Im MY, Kim SY (2002)
Arabidopsis basic leucine zipper proteins that mediate stress-responsive
abscisic acid signaling. Plant Cell
14:
343–357[Abstract/Free Full Text]
Karssen C (1995) Hormonal regulation of seed
development, dormancy, and germination studied by genetic control. In
J Kigel, G Galili, eds, Seed Development and Germination.
Marcel Dekker, Inc., New York, NY, pp
333–350
Kemp BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen Z-P,
Witters LA (1999) Dealing with energy demand: the
AMP-activated protein kinase. Trends Biochem Sci
24:
22–25[CrossRef][Web of Science][Medline]
Kim J, Harter K, Theologis A (1997)
Protein-protein interactions among the Aux/IAA proteins. Proc Natl Acad
Sci USA 94:
11786–11791[Abstract/Free Full Text]
Kim KN, Cheong YH, Grant JJ, Pandey GK, Luan S
(2003) CIPK3, a calcium sensor-associated protein kinase that
regulates abscisic acid and cold signal transduction in Arabidopsis.
Plant Cell 15:
411–423[Abstract/Free Full Text]
Kleinow T, Bhalerao R, Breuer F, Umeda M, Salchart K, Koncz
C (2000) Functional identification of an Arabidopsis
Snf4 ortholog by screening for heterologous multicopy suppressors of
snf4 deficiency in yeast. Plant J
23:
115–122[CrossRef][Web of Science][Medline]
Koornneef M, Bentsink L, Hilhorst H (2002) Seed
dormancy and germination. Curr Opin Plant Biol
5:
33–38[CrossRef][Web of Science][Medline]
Lakatos L, Klein M, Höfgen R, Bánfalvi Z
(1999) Potato StubSNF1 interacts with StubGAL83: a plant protein
kinase complex with yeast and mammalian counterparts. Plant J
17:
569–574[CrossRef][Web of Science][Medline]
Laurie S, McKibbin RS, Halford NG (2003)
Antisense SNF1-related (SnRK1) protein kinase gene represses transient
activity of an -amylase (-Amy2) gene promoter in
cultured wheat embryos. J Exp Bot
54:
739–747[Abstract/Free Full Text]
Liang P, Pardee AB (1992) Differential display
of eukaryotic messenger RNA by means of the polymerase chain reaction.
Science 257:
967–971[Abstract/Free Full Text]
Lopez-Molina L, Mongrand S, Chua NH (2001) A
postgermination developmental arrest checkpoint is mediated by abscisic acid
and requires the ABI5 transcription factor in Arabidopsis.
Proc Natl Acad Sci USA 98:
4782–4787[Abstract/Free Full Text]
Lopez-Molina L, Mongrand S, Kinoshita N, Chua NH
(2003) AFP is a novel negative regulator of ABA signaling that
promotes ABI5 protein degradation. Genes Dev
17:
410–418[Abstract/Free Full Text]
Lopez-Molina L, Mongrand S, McLachlan DT, Chait BT, Chua NH
(2002) ABI5 acts downstream of ABI3 to execute an ABA-dependent
growth arrest during germination. Plant J
32:
317–328[CrossRef][Web of Science][Medline]
Lu C, Han M-H, Guevara-Garcia A, Fedoroff NV
(2002) Mitogen-activated protein kinase signaling in
postgermination arrest of development by abscisic acid. Proc Natl Acad
Sci USA 99:
15812–15817[Abstract/Free Full Text]
Lumbreras V, Mar Albà M, Kleinow T, Koncz C, Page
M (2001) Domain fusion between SNF1-related kinase subunits
during plant evolution. EMBO Rep
21:
55–60[CrossRef]
Man AL, Purcell PC, Hannappel U, Halford NG
(1997) Potato SNF1-related protein kinase: molecular cloning,
expression analysis and peptide kinase activity measurements. Plant Mol
Biol 34:
31–43[CrossRef][Web of Science][Medline]
Muranaka T, Banno H, Machida Y (1994)
Characterization of the tobacco protein kinase, NPK5, a homologue of
Saccharomyces cerevisiae SNF1 that constitutively activates
expression of the glucose–repressible SUC2 gene for a secreted
invertase of S. cerevisiae. Mol Cell Biol
14:
2958–2965[Abstract/Free Full Text]
Nakamura S, Lynch TJ, Finkelstein RR (2001)
Physical interactions between ABA response loci of Arabidopsis.
Plant J 26:
627–635[CrossRef][Web of Science][Medline]
Nambara E, Hayama R, Tsuchiya Y, Mishimura M, Kawaide H, Kamiya
Y, Naito S (2000) The role of ABI3 and
FUS3 loci in Arabidopsis thaliana on phase transition from
late embryo development to germination. Dev Biol
220:
412–423[CrossRef][Web of Science][Medline]
Niu X, Helentjaris T, Bate NJ (2002) Maize ABI4
binds coupling element1 in abscisic acid and sugar response genes.
Plant Cell 14:
2565–2575[Abstract/Free Full Text]
Östling J, Carlberg M, Ronne H (1996)
Functional domains in the Mig1 repressor. Mol Cell Biol
16:
753–761[Abstract/Free Full Text]
Pien S, Wyrzykowska J, Fleming AJ (2001) Novel
marker genes for early leaf development indicate spatial regulation of
carbohydrate metabolism within the apical meristem. Plant J
25:
663–674[CrossRef][Web of Science][Medline]
Pritchard SL, Charlton WL, Baker A, Graham IA
(2002) Germination and storage reserve mobilization are regulated
independently in Arabidopsis. Plant J
31:
639–647[CrossRef][Web of Science][Medline]
Purcell PC, Smith AM, Halford NG (1998)
Antisense expression of a sucrose non-fermenting-1-related protein kinase
sequence in potato results in decreased expression of sucrose synthase in
tubers and loss of sucrose-inducibility of sucrose synthase transcripts in
leaves. Plant J 14:
195–202
Raz V, Bergervoet JHW, Koornneef M (2001)
Sequential steps for developmental arrest in Arabidopsis seeds.
Development 128:
243–252[Abstract]
Rolland F, Moore B, Sheen J (2002) Sugar
sensing and signaling in plants. Plant Cell
14:
S185–S205[Free Full Text]
Rook F, Corke F, Card R, Munz G, Smith C, Bevan MW
(2001) Impaired sucrose-induction mutants reveal the modulation
of sugar-induced starch biosynthetic gene expression by abscisic signaling.
Plant J 26:
421–433[CrossRef][Web of Science][Medline]
Rose JD, Winston F, Hieter P (1990)
Methods in Yeast Genetics: A Laboratory Course Manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schmidt MC, McCartney RR (2000) -Subunits
of Snf1 kinase are required for kinase function and substrate definition.
EMBO J 19:
4936–4943[CrossRef][Web of Science][Medline]
Slocombe SP, Laurie S, Bertini L, Beaudoin F, Dickinson JR,
Halford NG (2002) Identification of SnIP1, a novel protein
that interacts with SNF1-related protein kinase (SnRK1). Plant Mol
Biol 49:
31–44[CrossRef][Web of Science][Medline]
Smeekens S (2000) Sugar-induced signal
transduction in plants. Annu Rev Plant Physiol Plant Mol Biol
51:
49–81[CrossRef][Web of Science]
Söderman EM, Brocard IM, Lynch TJ, Finkelstein RR
(2000) Regulation and function of the Arabidopsis
ABA-insensitive4 gene in seed and abscisic acid response signaling
networks. Plant Physiol 124:
1752–1765[Abstract/Free Full Text]
Sugden C, Donaghy PG, Halford NG, Hardie DG
(1999) Two SNF1-related protein kinases from spinach leaf
phosphorylate and inactivate HMG-CoA reductase, nitrate reductase and sucrose
phosphate synthase in vitro. Plant Physiol
120:
257–274[Abstract/Free Full Text]
Takano M, Kajiya-Kanegae H, Funatsuki H, Kikuchi S
(1998) Rice has two distinct classes of protein kinase genes
related to SNF1 of Saccharomyces cerevisiae, which are
differently regulated in early seed development. Mol Gen Genet
260:
388–394[CrossRef][Web of Science][Medline]
Thornton C, Snowden MA, Carling D (1998)
Identification of a novel AMP-activated protein kinase subunit isoform
that is highly expressed in skeletal muscle. J Biol Chem
273:
12443–12450[Abstract/Free Full Text]
Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K,
Yamaguchi-Shinozaki K (2000) Arabidopsis
basic leucine zipper transcription factors involved in an abscisic
acid-dependent signal transduction pathway under drought and high-salinity
conditions. Proc Natl Acad Sci USA
97:
11632–11637[Abstract/Free Full Text]
Verhey SD, Walker-Simmons MK (1997) Abscisic
acid-mediated responses in seeds involving protein kinases and phosphatases.
In RH Ellis, M Black, AJ Murdoch, TD Hong, eds, Basic and
Applied Aspects of Seed Biology. Kluwer Academic Publishers,
Dordrecht, The Netherlands, pp
225–233
Vincent O, Townley R, Kuchin S, Carlson M
(2001) Subcellular localization of the Snf1 kinase is regulated
by specific subunits and a novel glucose signaling mechanism.
Genes Dev 15:
1104–1114[Abstract/Free Full Text]
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A. B. Downie, L. M. A. Dirk, Q. Xu, J. Drake, D. Zhang, M. Dutt, A. Butterfield, R. R. Geneve, J. W. Corum III, K. G. Lindstrom, et al.
A physical, enzymatic, and genetic characterization of perturbations in the seeds of the brownseed tomato mutants
J. Exp. Bot.,
May 1, 2004;
55(399):
961 - 973.
[Abstract]
[Full Text]
[PDF]
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