The Plant Cell, Vol. 13, 2687–2702, December 2001, www.plantcell.org © 2001 American Society of Plant Biologists
Tomato SP-Interacting Proteins Define a Conserved Signaling
System That Regulates Shoot Architecture and Flowering
Lilac Pnueli,a Tamar Gutfinger,a Dana Hareven,a Orna Ben-Naim,a Neta Ron,a Noam Adir,b and Eliezer Lifschitza,1
a Department
of Biology, Science and Technology, Technion, Israel Institute of Technology 32000, Haifa, Israel
of Chemistry and Institute of Catalysis, Science and Technology, Technion, Israel Institute of Technology
32000, Haifa, Israel
b Department
Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family
of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a
novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of
the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features
known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein–protein association domains of SP may be
separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.
INTRODUCTION
The shoot systems of flowering plants display great variation in their architecture and growth habit. The majority of
this variation can be attributed to modifications of the fundamental branching pattern (Bell, 1992). These modifications result from alternate states of the shoot apical meristem,
which can show either determinate or indeterminate growth,
and either vegetative or reproductive development.
Two model plant species, Arabidopsis and Antirrhinum,
have simple (monopodial) shoot architecture, that is, the
apical meristem is indeterminate and active throughout the
plant life cycle so that all appendages (leaves, side
branches, and floral buds) are clearly lateral. A decision to
flower is made only once during the life cycle of these species, after which the main shoot and axillary buds develop
reproductive organs. This results in a clear distinction between vegetative and reproductive phases.
In contrast, vegetative and reproductive phases alternate
regularly along the compound (sympodial) shoots of tomato.
The primary vegetative apex is terminated by an inflorescence after six to 20 leaves have formed, but upward
growth then continues from a new vegetative shoot arising
1 To
whom correspondence should be addressed. E-mail lifs@techunix.
technion.ac.il; fax 972-4-8225153.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.010293.
from the uppermost axillary bud just below the terminating
inflorescence. From then on, the stem is composed of reiterated units, each with three nodal leaves and a terminal inflorescence. Termination of each vegetative apex is thus
synonymous with the transition to flowering in tomato but
not in Arabidopsis or Antirrhinum. These basic differences in
the meristematic environments are reflected in the changes
incurred by homologous mutations on the overall architecture of the two plant species (Pnueli et al., 1998).
Mutations in the TERMINAL FLOWER1 (TFL1) gene of Arabidopsis or in the CENTRORADIALIS (CEN) gene in Antirrhinum (Shannon and Meeks-Wagner, 1991; Alvarez et al.,
1992; Bradley et al., 1996, 1997) convert the inflorescence
apical meristem from a normally indeterminate to a determinate state after a few flowers are formed. This results primarily in a shorter inflorescence shoot bearing a terminal
flower. Yet an altered termination pattern in tomato results
in an overall dramatic change in the plant architecture. A recessive mutation in the SELF-PRUNING (SP) gene (Yeager,
1927; MacArthur, 1932; Pnueli et al., 1998) confers accelerated termination of stem units until the shoot is eventually
terminated by two consecutive inflorescences. Furthermore,
in contrast to mutations in the CEN and TFL genes, sp mutation is inconsequential for the architecture of the inflorescence itself, because, unlike that in Arabidopsis, the
inflorescence in tomato is inherently determinate (Pnueli et
al., 1998). The “determinate” habit of the main shoot of tomato is repeated in side shoots, resulting in a limited growth
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of each shoot, a bushy compact constitution, and nearly homogeneous fruit setting. Introduction of the recessive sp
gene into tomato cultivars revolutionized the tomato industry because the determinate growth habit facilitates mechanical harvesting (Atherton and Harris, 1986). Determinate
sp plants also display a range of pleiotropic effects: internodes are shorter, more flowers are formed per inflorescence, control of apical dominance is relaxed, seed
germination is accelerated, and plants are more prone to
auxin treatment. In wild-type plants, auxin and its synergists
induce determinacy and other characteristics of the sp phenotype (Zimmerman and Wilcoxon, 1942; Zimmerman and
Hitchcock, 1949; Teubner and Wittwer, 1957; Gardner and
Hedger, 1959; Lifschitz, 1965). In addition, the phenotypic
expression of sp varies considerably in different genetic
backgrounds (Pnueli et al., 1998), suggesting to us that the
function of the SP gene is directly, or indirectly, mediated by
auxin.
The CEN, TFL, and SP genes have been cloned and
shown to be closely related (Bradley et al., 1996, 1997;
Pnueli et al., 1998). Thus, although homologous gene mutations have different consequences in different shoot systems, in each case it seems that these genes determine the
potential for continuous growth of the shoot apical meristem. This is because contrary to the mutant effect, overexpression of CEN, TFL1, or SP prolongs the vegetative stage.
In extreme cases, expression of CEN (Amaya et al., 1999) or
SP in tobacco (L. Pnueli and E. Lifschitz, unpublished data)
may delay the transition to flowering for years. Interestingly,
another gene of Arabidopsis, FLOWERING LOCUS T (FT),
also has been shown to be a member of the same gene
family (Kardailsky et al., 1999; Kobayashi et al., 1999). The
product of this gene is apparently antagonistic to that of TFL1
in that overexpression of FT mimics the loss of TFL1 function,
and vice versa (Koornneef et al., 1991; Ratcliffe et al., 1998).
CEN/TFL/SP and FT are members of a small gene family,
with approximately six members in tomato (L. CarmelGoren, personal communication) and six in Arabidopsis (D.
Weigel, personal communication), that encodes 23-kD proteins. They share sequence similarity with a group of mammalian polypeptides designated phosphatidylethanolamine
binding proteins (PEBPs; Grandy et al., 1990; Schoentgen
and Jolles, 1995). The ability of PEBPs to bind phospholipids in vitro promoted the suggestion (Bradley et al., 1997)
that they play a role in signaling. The crystal structures of
PEBP from human and bovine sources (Banfield et al., 1998;
Serre et al., 1998) and that of the CEN protein from Antirrhinum (Banfield and Brady, 2000) have been determined.
Structural analysis of CEN suggests that its ligand binding
pocket is incapable of accommodating phospholipid and
thus is unlikely to function via direct contact with lipid bilayers. It is capable of accommodating phosphoryl groups,
however, suggesting that PEBP proteins may mediate signaling via their association with phosphorylated proteins. Indeed, a mouse PEBP, RKIP, was recently shown to interact
with and inhibit activity of the kinase Raf1 (Yeung et al.,
1999, 2000). Rather than using the inappropriate designation PEBP, we suggest that this gene family be named the
CETS genes after the first three plant genes with identified
biological functions—CEN, TFL1, and SP.
CETS genes play a critical role in shaping plant architecture that is conserved among species. CETS genes do not,
however, encode proteins that belong to families of DNA
binding proteins, transcription activators, kinases, or receptors that are known to regulate major developmental programs in plants. CETS genes have no effect on cell survival,
cell fate, or organ identity. We believe that in meristems,
they decide the timing of potential switching from vegetative
to reproductive growth.
To understand the molecular function of this new group of
plant regulatory factors, we have initiated the identification
and isolation of SP-interacting proteins (SIPs). It is believed
that the combination of mutant phenotypes, interacting proteins, and resolved crystal structures will lead eventually to
a comprehensive understanding of the mechanisms that facilitate the reproductive, species-specific architecture of
flowering plants.
RESULTS
Molecular Identity of the SIPs Suggests a Role for SP in
Molecular Signaling
The full-length cDNA of the SP gene was used as bait to
screen 106 clones of an activation-domain fusion library prepared from RNA of shoot apices of wild-type tomato (see
Methods). The scale is not exhaustive, and thus far five pu-
Table 1. Characteristics of SIPs
Number
Type
Two Hybrida
cDNAa
Locusb
SIP2
SIP74
SIP3
SIP4
SIP8
14-3-3/2
14-3-3/74
SPAKc
Novel
SPGBd
258
252
339,220
98
62
258
252
609
99
NDe
11-1, 11-2
4-3
2-1, 2-2 (?)
11-1
2-4, 2-5
a Length
(amino acids).
assignments were obtained using 50 tomato lines containing overlapping insertions of the heterologous Lycopersicon pennellii
wild species (Eshed and Zamir, 1995). SIP2, for example, was
mapped to a region on chromosome 11 shared by insertions 11-1
and 11-2. The question mark in the last column indicates that the
mapping of SIP3 to IL2-2 was not conclusive. Only clones that were
identified in the primary screen, using SP as a bait, are included in
the table.
c SPAK, NIMA-like kinase.
d SPGB, SIP8.
e ND, not determined.
b Linkage
Self-Pruning Interacting Proteins
tative SIPs were identified. Their basic characteristics are
shown in Table 1.
SIP2 and SIP74 (Table 1) represent isoforms of the 14-3-3
family. One other interacting member of the 14-3-3 family,
14-3-3/5, is not included in Table 1 because it was isolated
in a subsequent, limited, two-hybrid screen using the
shorter SIP3 clone (Figrure 1A) as bait, and it has not been
genetically mapped. SIP2 is a novel isoform of 14-3-3, and
SIP74 represents the epsilon isoform. Members of this family are referred to in this article as 14-3-3/2, 14-3-3/74, and
14-3-3/5, respectively. 14-3-3s are a class of adapter proteins involved in signaling, transcription, and compartmentalization via their ability to stabilize, dimerize, or bridge their
substrates, for example, Raf1, Cdc25, and BAD (Aitken,
1996; Muslin et al., 1996; Zha et al., 1996; Peng et al., 1997;
Roberts et al., 1997; Brunet et al., 1999; Lopez-Girona et al.,
1999; Pan et al., 1999; Yang et al., 1999).
SIP3 is a serine/threonine kinase, designated SPAK (for
SP-associated kinase). SPAK shares 60 to 65% similarity,
in its catalytic domain, with the NIMA-like kinases (for never
in mitosis A). Two independent clones extending for different lengths to the N terminus were isolated (Table 1). A fulllength cDNA, encoding 609 amino acids, was isolated subsequently from our regular cDNA library. Six related sequences were identified in the Arabidopsis sequence
database. The NIMA kinase regulates early and late progression of G2 stages in Aspergillus and mammalian cells
and is required for entry into mitosis and for the nuclear localization of the cyclin B/cdc2 complex (Osmani et al., 1988,
1991; Fry and Nigg, 1995; Lu et al., 1993; Lu and Hunter,
1995; Ye et al., 1995; Wu et al., 1998). It has been suggested (Cortez and Elledge, 2000) that the NIMA kinase may
serve as the target for Chfr, a newly identified mitotic checkpoint protein regulating chromosome condensation (Scolnick
and Halazonetis, 2000). SIP4 is a short, 10-kD (99–amino
acid) novel protein. It is related to two anonymous genomic
sequences from the Arabidopsis genome project. SIP8,
named SPGB, is a putative bZIP transcription factor, a subclass of G-box (CCACGTGG) binding proteins (Giuliano et
al., 1988; Menkens et al., 1995). It has been shown that G-box
factors are phosphorylated before occupying their position in
the transcription complex (Harter et al., 1994) and that 14-3-3
isoforms are components of transcription complexes containing G-box binding factors (Lu et al., 1992). On the basis of
the 62 C-terminal amino acids sequence currently available,
the family member most similar to SPGB is GBF4 (Menkens
and Cashmore, 1994).
Tomato SIPs Bind to CETS Proteins from Arabidopsis
and Antirrhinum
To explore the relevancy of the SP/SIPs associations, we
have investigated whether their interactions are conserved
in other species (Figure 1A). The CEN gene of Antirrhinum
and TFL1 of Arabidopsis are orthologs of SP (Pnueli et al.,
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1998) and maintain, in their respective species, the indeterminate state of the inflorescence meristem (Amaya et al.,
1999). The determinate phenotype in tomato is complemented by CEN (Pnueli et al., 1998), and SP, like CEN, delays flowering in tobacco plants; it also induces a leafy-like
phenotype in tfl1 mutant plants of Arabidopsis (results not
shown). FT is a functionally divergent CETS protein from Arabidopsis that apparently plays a role antagonistic to that of
TFL1 (Kardailsky et al., 1999; Kobayashi et al., 1999). In addition to these three homologs, we have tested three overlapping peptides of SP spanning residues 1-42, 38-95, and
43-175. They are collectively denoted, in the second column
of Figure 1A, by a SP. None of the five SIPs bound to any
of the truncated SP proteins. In contrast, both CEN and
TFL1 bind SPAK, 14-3-3/74, and the SPGB proteins but do
not react significantly with SIP4. FT, the functional antagonist of TFL1, displays the same binding pattern as TFL1.
Thus, some of the SIP interactions in tomato are apparently
conserved in distantly related plants. The interactions of FT
with SIPs, however, do not provide a molecular basis for its
antagonistic role in flowering.
SIPs Form a Network of Bipartite Interactions
The molecular identity of the SIPs supports their involvement in signaling processes. To test the possibility that they
are involved in a SP-dependent signaling network, we examined them for their ability to form associations among
themselves. Three 14-3-3s were included in some of the
pairwise tests to probe possible differential affinities with the
other SIPs or among themselves. Results of the two-hybrid
tests are shown in Figures 1B and 1C (and supporting in
vitro assays are discussed in relation to experiments documented in Figure 3).
Results of survival assays carried on the histidine ( His)
selective medium (see Methods) suggest that SPAK, SIP4,
and 14-3-3/74 may form homodimers in yeast cells (Figure
1B). SP, by contrast, does not form homodimers (data not
shown), which is consistent with the conclusion derived
from the structural analysis of CEN (Banfield and Brady,
2000). Evidently, 14-3-3 isoforms, initially recovered as interacting with SP, also interact with SPAK and SIP4. Because G-box binding proteins are known to interact with 143-3s, the lack of interactions between 14-3-3s and the
SPGB is attributed to the short fragment of the gene available to us. Presumably, SP and 14-3-3s are associated with
separate domains of the GB protein.
Corroborating -galactosidase assays are documented in
Figure 1C. Interactions involving SPGB were omitted from this
test because they were negative in the much more sensitive
assay shown in Figure 1B. Strong interactions occur between SPAK and 14-3-3/74, and between SIP4 and 14-3-3/
74. SPAK and SIP4 show weaker, albeit differential, interactions with 14-3-3/5 and 14-3-3/2. Interestingly, 14-3-3/74,
which weakly dimerizes, forms a very strong association
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with 14-3-3/2 and only slightly less so with 14-3-3/5; these,
in turn, are the weakest interactors in other combinations.
Such differential affinities may form a basis for subtle modifications of the SP system by 14-3-3 factors.
Expression Domains of SPAK, SPGB, and SIP4 Overlap
with That of SP
Expression domains of three SIP genes were compared, by
in situ hybridization, with the expression pattern of SP.
SPAK is expressed from the beginning in the apical meristem and in the leaf and stem vasculature of the primary
apex of a young, two-leaf seedling (Figure 2A). A similar pattern is observed in the floral primordia of the primary inflorescence (Figure 2B) and in the vegetative meristem of the
first sympodial segment (Figure 2B). In the developing floral
bud, SPAK expression is particularly prominent in the stamens and carpels (Figure 2C). This expression pattern of
SPAK overlaps spatially and temporarily with the patterns
observed for SP and for the tomato LEAFY gene (Pnueli et
al., 1998).
The in situ localization of transcripts of the SPGB and
SIP4 genes is shown only for the apical meristems of young
seedling (Figures 2D and 2E) because they too overlap
throughout with that of SPAK and SP. In addition, the same
pattern was seen for 14-3-3/2 (data not shown). Because all
three probes shown in Figures 2A to 2E, along with SP and
the T-Leafy probes, mark the same meristematic domains,
we have used the tomato ribonucleotide-reductase small
subunit gene (RNR2; E. Lifschitz and M. Egea-Cortines, unpublished data) as a positive control probe. This gene is expected to be expressed at high levels in meristematic
tissues because it is upregulated specifically during S-phase;
thus, it displays a noncontinuous, salt-and-pepper pattern
(Figure 2F).
SP Interacts in Vitro with SIPs
In vitro assays were performed to examine the ability of the
SIPs to associate with SP and among themselves, independent of potential yeast partners. A FLAG-SP fusion protein,
expressed in Sf9 insect cells, was tested in pairwise combinations with radiolabeled 14-3-3/74, SPAK, and SIP4 preFigure 1. Identification and Characterization of the SIPs in the Yeast
Two-Hybrid System.
(A) Conserved interactions between SIPs and SP homologs from Arabidopsis and Antirrhinum. Results of the two-hybrid tests between
CETS and SIPs are illustrated. The FT column was inserted from a
separate plate because BD-FT displays a residual growth (meaning
activation potential) on the His selective medium, thus requiring
tests in plates supplemented with 4 mM triamino triazole (3AT). SP
refers collectively to three overlapping peptides of SP (see text), all
of which gave the same negative result.
In (B) and (C), 14-3-3 proteins interact with all other SIP proteins.
(B) Bipartite associations between SIP proteins in a survival, twohybrid test, on the His selective medium, are shown.
(C) Colony lift, -galactosidase association assays between SIPs
are shown.
The strength of the interactions ranges from 11 Miller units for the
SPAK:14-3-3/74 pair to 0.4 units for SPAK:14-3-3/2. Note that for
the presence of positive interactions, a positive control between p53
and SV40 is not included here; P53 is used as a negative control.
Self-Pruning Interacting Proteins
2691
Figure 2. Expression Domains of SPAK, SIP4, and SPGB Overlap with That of SP.
(A) to (C) Localization of SPAK mRNA in the early (two-leaf stage) vegetative apex (A), the first sympodial apex and an early floral primordium
(B), and a floral bud (C).
(D) and (E) Localization of SIP4 (D) and SPGB (E) mRNA in early vegetative apices.
(F) Control section displaying contrasting expression pattern of the gene for the small subunit of the tomato ribonucleotide-reductase small subunit gene (RNR2). RNR2 displays a discontinuous pattern, reflecting the distribution of cells in S phase.
Digoxygenin-labeled antisense RNA probes for in situ hybridizations were prepared from inserts cloned in the BS vector using the T7 polymerase. C, carpel; FP, floral primordium; L, leaf; P, petal; PR, primary apex; S, sepal; SA, sympodial apex; ST, stamen; VS, vascular strand.
pared in the in vitro transcription/translation system and, as
shown in Figure 3A, sections I to III, interacts readily with all
three tested proteins. Identical results were obtained with
FLAG-SP expressed in yeast cells (results not shown). In
vitro binding assays shown in Figure 3A, section IV, verified
also the in vitro association between SPAK and 14-3-3/74,
the lack of association between SPAK and SIP4, and the
potential of SPAK to dimerize. The results of the in vitro
studies thus are consistent with the results of the two-hybrid
experiments illustrated in Figure 1.
SPAK and 14-3-3/74 Interact in a
Phosphorylation-Dependent Manner
For the in vitro reciprocal binding assays between pairs of
SIPs, we used GST fusion proteins expressed in bacteria
and 35S-methionine–labeled proteins translated in vitro. In
preliminary experiments, some pairwise reciprocal assays
did not give consistent results. It was subsequently determined that SPAK and SIP4 (used, for example, in the experiments described in Figure 3A), but not 14-3-3/74, are
actually phosphorylated by kinases present in the in vitro
translation system.
Therefore, for the initial evaluation of the role of phosphorylation in interactions between SIP proteins, the possibility
that SPAK is autophosphorylated, and that it phosphorylates other SIP proteins, was investigated. For autophosphorylation, we tested the ability of full-length SPAK to
phosphorylate a truncated protein deleted for its catalytic
domain (amino acids 1 to 270; see Figures 4A to 4C). As
shown in Figure 3B, secton I, lanes 1 and 2, the deleted
SPAK protein is indeed phosphorylated by full-length SPAK
expressed in bacteria. Of the other SIPs, only SIP4 was
phosphorylated, in vitro, by SPAK (Figure 3B, section I, lane
3), thus establishing a biochemical function for SPAK. Deletion analysis of SIP4 suggested that the phosphorylation
site was present in the C-terminal 20 amino acids (Figure
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3B, section I, lanes 4 and 5), where the only candidate is a
serine 98, embedded in a potential phosphorylation domain
(R/KXXS/T).
Because in many cases 14-3-3 binds phosphorylated
proteins (Yaffe et al., 1997b; Finnie et al., 1999), the requirement for SPAK and SIP to be phosphorylated to associate
with 14-3-3/74 was examined. Pull-down assays shown in
Figure 3B, sections II and III, revealed that in vitro–translated
14-3-3/74, which by itself is not phosphorylated, binds only
phosphorylated SPAK (Figure 3B, section II); however, to
form homodimers, 17-3-3/74 needs not to be phosphorylated. Likewise, it was found that only a phosphorylated
SIP4 binds 14-3-3/74 (Figure 3B, section III).
SP and 14-3-3/74 Share a Six–Amino Acid Binding
Domain in the SPAK Protein
The results described thus far show that SPAK binds to
both SP and 14-3-3 proteins. Another serine/threonine kinase, Raf1, provides a precedent because it is associated
with 14-3-3 (Rommel et al., 1996; Muslin et al., 1996; Yaffe
et al., 1997a; Rittinger et al., 1999) and with a mammalian
homolog of SP, RKIP (Yeung et al., 1999, 2000). The interactions of 14-3-3 and SP with SPAK were therefore studied
in more detail. The amino acid sequence of SPAK is shown
in Figure 4A. The deduced catalytic and dimerization domains, and the binding sites for SP and 14-3-3/74, are
shown schematically in Figure 4B. Deletion analysis in the
yeast two-hybrid system suggests that the C-terminal do-
cells were immobilized on anti-FLAG agarose beads, incubated with
SIPs, resolved on SDS-PAGE, and visualized by autoradiography. In IV, SPAK polypeptides form homodimers and interact in vitro with 14-3-3/74 but not with SIP4. Note that SPAK in
IV, as well as SPAK and SIP4 in II and III, respectively, are phosphorylated.
(B) Phosphorylation-dependent associations between SIP proteins.
In I, SPAK auto-phosphorylates and also phosphorylates the C-terminal region of SIP4. A GST fusion with the complete SPAK protein
was incubated in a phosphorylation reaction assay with a GST control (lane 1); a GST-SPAK 270-609 protein lacking the kinase domain
(60 kD; see Figure 6) (lane 2); a GST-SIP4 fusion protein (36 kD;
amino acids 1 to 99) (lane 3); a GST-SIP4 76-99 (30 kD) (lane 4); and
a GST-SIP4 1-75 N-terminal domain (lane 5). Proteins were separated by SDS-PAGE and autoradiographed. In II, 14-3-3/74 dimerizes
and interacts with phosphorylated (*), but not with nonphosphorylated, GST-SPAK fusion protein. In III, 14-3-3/74 protein binds to
GST-SIP4 only if the latter is phosphorylated. In both II and III, in
vitro–translated 35S-Met–labeled SIPs (indicated to the right of each
gel) were incubated with immobilized GST-SIP fusion proteins (indicated above each gel). The asterisks indicate a GST-SIP previously
phosphorylated by SPAK. Samples of bound radioactive proteins
were eluted, resolved on SDS-PAGE, and visualized by autoradiography.
35S-Met–labeled
Figure 3. SP and SIPs Interact in Vitro.
(A) In I to III, in vitro association of labeled 14-3-3/74, SPAK, and
SIP4, respectively, to FLAG-SP protein is shown. The FLAG-SP gene
was expressed in Sf9 cells. Proteins from expressing cells and control
Self-Pruning Interacting Proteins
2693
Figure 4. SP and 14-3-3 Share the Same Binding Site in SPAK.
(A) Amino acid sequence of the SPAK protein. Some conserved amino acids characteristic of the catalytic domain of kinases are marked in blue.
The two putative 14-3-3 binding sites are shown in boldface and red, respectively. Serine 406 of the C-terminal site shared by 14-3-3 and SP is
indicated by an asterisk.
(B) Schematic representation of the major functional domains in the SPAK protein. The scheme is based on data derived from sequence comparisons and deletion analysis in the two-hybrid system.
(C) Identification of the binding region for Sp and 14-3-3. Fragments of the regulatory domain of SPAK were tested in yeast for their interaction
with binding domain fusions of SP, the SPAK regulatory domain (amino acids 270 to 609), and 14-3-3/74. The black bars at right show deletions
of SPAK used to map the dimerization domain and the SP/14-3-3 binding region. The three columns at center show the results of the yeast twohybrid assays. The ability () or the failure () to survive on His selective medium is indicated. Note that binding of SP and 14-3-3 to SPAK is effective only in the presence of the dimerization domain (amino acids 511 to 609).
(D) Fine-scale mapping of the 14-3-3 and SP binding site within SPAK. The amino acid sequence of the putative wild-type SP/14-3-3 binding region (389 to 417) of SPAK is shown. The consensus sequence for the binding of 14-3-3 (Yaffe et al., 1997a) and the conservative serine 406 are
highlighted in red. Delineated below are the sequences of the three mutant versions of SPAK 389-417 that were used for the detailed mapping
of the common binding site. The first retained the consensus six–amino acid sequence at its N terminus (I), the second polypeptide had this sequence deleted (II), and the third included the 14-3-3 consensus sequence but with serine 406 replaced by alanine (III). Results of two-hybrid
tests of interactions between these modified SPAK proteins with SP, SPAK, and 14-3-3/74 are shown at bottom. Serine 406 is required for the
binding of both SP and 14-3-3 under these conditions (plate at the bottom right).
main of SPAK, amino acids 511 to 609, is required for homodimerization (Figure 4C). A binding site for SP must also
lie within the larger 389 to 609 region because it alone was
present in the smaller of the two original cDNA products that
bound SP (Table 1). A deletion analysis of SPAK also reveals
that a 29–amino acids long peptide, spanning positions 389
to 417, inclusive, is required for the binding of both SP and
14-3-3/74, but binding is effective only in conjunction with a
C-terminal dimerization domain (Figure 4C).
The sequence of the putative, 29–amino acid long, binding domain for SP and 14-3-3/74 contains a classical consensus sequence for the binding of 14-3-3 proteins (Yaffe et
al., 1997a; Figures 4A and 4D), raising the unexpected possibility that SP and 14-3-3 share the same binding site
within SPAK.
To address this possibility, we further dissected the 389to-417 peptide of SPAK 389-609 into three subregions (Figure 4D) and tested the mutated SPAK proteins against SP
and 14-3-3/74 in the yeast two-hybrid system. The results
narrowed the essential SPAK site to the six–amino acids sequence (shown in red) that forms the consensus for the
binding of 14-3-3 proteins. Included in this peptide is a conservative serine residue in position 406 that is likely to be
crucial for effective binding with 14-3-3/74 (Yaffe et al.,
1997b; Rittinger et al., 1999). Indeed, replacement of serine
406 by alanine (Figure 4D, lines III) abolished the binding of
SPAK 389-609 to 14-3-3/74 and to SP but not the formation
of SPAK dimmers, thus providing further evidence that SP
and 14-3-3/74 may be interchangeable at, or even compete
for, this SPAK site under certain physiological conditions.
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In Raf1, two 14-3-3 molecules form a bivalent intramolecular bridge connecting binding sites in its N-terminal,
RSTS259TP and C-terminal, RSAS621EP domains (Li et al.,
1995; Rommel et al., 1996; Muslin et al., 1996). Likewise
there is, in addition to the serine 406 site, another potential
binding site for 14-3-3, RRNS274LP, in the N-terminal domain of SPAK. To examine the binding potential of this second site to 14-3-3/74, a full-length SPAK that includes the
S274 site but with S406 replaced by alanine was examined.
The full-length SPAK S406A retained its binding to 14-3-3,
but no binding to SP was detected (results not shown),
which perhaps implies that SPAK has only one potential site
for SP but, like Raf1, at least two potential binding sites for
14-3-3.
Overexpression of 14-3-3 Genes Compensates for the
Loss of Function of SP
To explore the functional role of the SIP genes in the context
of plant architecture, we have initiated their study in transgenic plants. At first, the 14-3-3 and SPAK genes were used
to transform plants of the determinate double mutant sp an
(anantha; Paddock and Alexander, 1952) genotype because
anantha plants are more sensitive to the growth-promoting
effects of the SP gene in two ways (Pnueli et al., 1998). First,
the determinate habit of the sp;an shoots (Figure 5B; cf. Figure 5A) is converted by the overexpression of SP to a highly
irregular, indeterminate pattern, with three to six internodes
between inflorescences (Figure 5C). Second, the primordia
of the sp;an inflorescence, which are normally arrested at a
pre-floral, cauliflower-like state (Figure 5E), now develop
leaflets and leaves (Figures 5C and 5F). Thus, transgenes affecting the SP system are expected foremost to alter the determinate habit of sp;an shoots and/or the stage in which
the primordia of the anantha inflorescence are arrested.
No developmental changes were observed in sp or sp;an
plants expressing antisense RNA of the 14-3-3/2 or 14-3-3/
74 genes. However, a clear manifestation of increased vegetative characteristics is evident in the shoots and inflorescence of sp;an plants overexpressing either of the two
genes (Figures 5D, 5G, and 5H). A significant attenuation of
the determinate phenotype was observed in sp;an plants
overexpressing 14-3-3/2 (Figure 5D), although this was not
as complete as observed when SP is overexpressed in the
same background (Figure 5C). The now indeterminate main
and side shoots of sp;an plants expressing 14-3-3/2 feature
a variable number of one to three internodes between inflorescences, with spacings of one or two leaves predominating. In addition, the inflorescence is now mildly leafy (Figure
5G). Overexpression of 14-3-3/74 in sp;an plants resulted in
only mild, albeit consistent, delay of determinacy, so that
many branches generated five to seven inflorescence
shoots before termination, as compared with three to four
inflorescence shoots in the progenitor line. However, the
second growth-promoting parameter, that is, the leafiness
of the inflorescence, is much more pronounced (Figure 5H).
Thus, increased levels of 14-3-3 proteins seem to compensate for the loss of function of the SP gene, at least in the
anantha mutant background, suggesting perhaps some
overlapping biochemical functions. It should also be noted
that the partial suppression of the determinate sp mutant
phenotype that is a semi-determinate habit is also characteristic of several genetic backgrounds such as that of the
VF36 line. Modifiers may include variants of SP-interacting
genes such as 14-3-3.
It will be interesting to see if overexpression, in the same
plants, of 14-3-3/2 and 14-3-3/74, which form strong heterodimers (Figure 1), will result in a more complete complementation of the determinate phenotype.
Antisense expression of SPAK has no obvious effect on
growth habit. However, all sp plants expressing antisense
RNA form pear-shaped fruits instead of the normal round
fruits of the progenitor plants. Pear-shaped, ovate fruits are
formed in transgenic plants bearing regular-size fruits as
well as in plants bearing cherry fruits (Figures 5I and 5J). Exactly the same results were obtained when a dominant-negative form of SPAK (i.e., amino acids 403 to 609) was
expressed in tomato plants. Outcrossing of antisenseexpressing plants with different determinate and indeterminate lines showed that formation of ovate fruits is independent of allelic variation in the SP locus itself.
Mutations in Conserved Structural Domain Annul the
SP/SIPs Interactions
To substantiate a functional need for SP to interact with
SIPs, we took advantage of the recently determined crystal
structures of three CETS (PEBP) proteins, from mammals
and plants (Banfield et al., 1998; Serre et al., 1998; Banfield
and Brady, 2000), and of single amino acid alterations
known to inactivate plant CETS genes (Bradley et al., 1996,
1997; Pnueli et al., 1998; Kardailsky et al., 1999; Kobayashi
et al., 1999). In principle, it should be possible, under these
circumstances, to relate binding potential of mutant SP variants to conformational features, known to be conserved, in
this new family of regulatory factors.
For a rational choice of mutant sites to be tested, we have
built a structural model of SP, using homology-based modeling (Peitsch, 1996). The resulting three-dimensional model
(see Figure 7A) is very similar to the structures reported for
PEBP proteins. We have subsequently analyzed the binding
properties of SP proteins carrying three known mutations in
particularly critical and conservative domains of all CETS/
PEBP proteins.
In the recessive sp allele, proline in position 76 (Pnueli et
al., 1998), designated here as P74 to conform with the alignment of all the genes in the family (Banfield and Brady,
2000), is replaced by leucine. Proline 74 is within an invariant motif, DPDXP74D, near the putative active site (indicated in Figure 7A by an oval frame and the position of H86),
Self-Pruning Interacting Proteins
2695
Figure 5. Overexpression of 14-3-3 genes in Determinate anantha Mutant Plants Mimics the Effect of Upregulation of SP.
(A) An indeterminate wild-type shoot. Four successive inflorescences spaced by three leaves each are indicated by arrows.
(B) A shoot from a progenitor determinate double mutant sp an plant. The shoot is terminated by two consecutive inflorescences (upper two arrows), which is the consequence of the sp mutation. The meristems in each inflorescence (arrows) proliferate, and all are arrested in a cauliflower-like stage (the effect of an).
(C) Overexpression of the SP gene in sp an mutant background. Note the increased number of leaves between inflorescences and the generation of leaves by the sp;an inflorescence meristems (arrows).
(D) Overexpression of the 14-3-3/2 gene results in the partial rescue of the mutant determinate habit of sp;an plants. Note the indeterminate pattern of the shoot (seven inflorescences are marked by arrows), and the two to three nodal leaves separating the inflorescence shoots.
(E) A single inflorescence of a progenitor, double mutant sp;an mutant. Meristems are arrested at the cauliflower-like stage, with no leafy primordia formed.
(F) Vegetative growth, that is, production of leaves in the sp;an mutant inflorescence of a plant overexpressing SP.
(G) Promotion of vegetative growth, that is, production of leaf primordia in the inflorescence of a sp;an mutant plant overexpressing 14-3-3/2.
(H) Leafy phenotype of the inflorescence of a sp;an mutant plant overexpressing 14-3-3/74.
(I ) and (J) Suppression of SPAK induces fruit elongation. (I) A regular size fruit of the VF36 line (right) and an elongated, pear shape fruit from
plants expressing p35S::SPAK antisense RNA. (J) Round cherry tomato fruits (left) and elongated cherry fruits (right) from plants expressing a
dominant-negative form of SPAK.
2696
The Plant Cell
which is conserved in all members of the family (Serre et al.,
1998; Banfield and Brady, 2000). Because no other mutant
alleles of SP are known, we have exploited two other conserved mutant sites of its homolog, the TFL1 gene of Arabidopsis (Bradley et al., 1997). One disfunctional allele of TFL1
(i.e., a terminal flower and early flowering) carries a threonine-to-isoleucine alteration in position 65 and the other a
glycine-to-aspartic acid in position 100 (Bradley et al.,
1997). These two sites are more distant from the ligand
binding site, and it appears that neither mutation potentially
alters SP ligand binding.
To investigate the effect of these mutations on the binding
properties of SP, we introduced identical mutations into the
SP gene. A change in one base was sufficient for the T65I
alteration, but two steps were required (G to E and E to D) to
introduce the G100D change in the SP. The three mutant
forms of SP were subsequently tested in yeast for their interaction with the SIP proteins. As shown in Figure 6, the
four SIPs interact readily with the SP-P74L mutant protein.
In contrast, with the exception of the successful binding between SP T65I and the short peptide representing the SPGB
protein, the SP-T65I, SP-G100E, and the SP-G100D mutants, as well as the double mutant SP-T65I G100E, abolished the SP–SIPs interactions in the two-hybrid test.
DISCUSSION
SPAK, SPGB, and 14-3-3s belong to protein families known
to be involved in signaling pathways; the novel SIP4 may be
Figure 6. Binding Properties of T65I and G100D Mark Putative Conservative Protein–Protein Association Domains of SP.
SP and its mutant derivatives used in this experiment were tagged
with FLAG (to verify expression in yeast) and fused to the Gal4 binding domain. The SP bait (double, in the far right column) denotes the
double mutant SP T65I:G100E.
phosphorylated by SPAK; and SP and the other SIPs all interact with 14-3-3s. The observed network of specific interactions between SIPs provides support for the assertion
that the SIPs are legitimate components of a putative SPdependent signaling system. Upstream effectors and downstream targets for the SP system are yet to be discovered,
and the full range of the biochemical diversification of CETS
proteins has not yet been explored. Even so, it is already
possible at this point to discuss the possible biochemical
function of SP in conjunction with mutations that interfere
with its binding properties and in relation to its interactions
with the 14-3-3 and SPAK proteins.
Molecular Interactions between SP, SPAK, and 14-3-3s
Suggest Analogy with the Raf1 Mechanism
The SP system shares notable similarities, with possible farreaching implications, with the Raf1 complex. Because
none of the known SP partners seems likely to anchor the
system to the membrane, the SP signaling system, like that
of Raf1, is more likely to be a transient component of membrane-associated effector complexes. Although Raf1 and
SPAK belong to two different families of kinases, they each
bind to members of the same protein family: SP, TFL1, and
CEN bind to SPAK, and RKIP, an SP homolog protein from
mammals, binds to Raf1 (Yeung et al., 1999, 2000). Both SP
and RKIP bind only phosphorylated kinases, the two kinases bind 14-3-3s in a phosphorylation-dependent manner, and each has two 14-3-3 binding sites. In fact, we
found that the NIMA kinase itself also contains two potential
binding sites for 14-3-3 proteins. In this context, it is noteworthy that if Raf1, like SPAK, also uses the 14-3-3 binding
site to attract the mammalian SP homolog RKIP, the analogy is extended and an unexpected additional regulatory
point for Raf1 is envisaged.
Here, we report that binding of SP to SPAK depends on a
serine residue at position 406. The phosphorylation status
of this particular serine has not been directly established,
but because the very same site is also required for the binding of 14-3-3/74, and because the requirement for a phosphorylated serine in the 14-3-3 class of binding sites has
already been established (Muslin et al., 1996), it is likely that
serine 406 of SPAK must be phosphorylated for SPAK to
bind SP as well. It is not implied, however, that the phosphoryl-serine 406 is actually accommodated by the ligand
binding pocket of SP.
In Raf1, two 14-3-3 molecules form bivalent intramolecular bridging sites in the N- and C-terminal domains before
being displaced from the N-terminal site by an activated
Ras (Li et al., 1995; Rommel et al., 1996; Muslin et al., 1996).
Likewise there are two binding sites for 14-3-3 in SPAK, but
it seems as if only one is shared by SP. Exclusive SP
bridges in SPAK are currently excluded also because SP
does not dimerize. However, potentially SP could participate
in an intramolecular bridge in cooperation with a 14-3-3 if
Self-Pruning Interacting Proteins
2697
transient modifications of SPAK by other accessory proteins
occur. Pin1, a peptidyl-prolyl isomerase, is one possible
agent for such a structural modification of SPAK. Pin1 was
initially identified as interacting with, and inhibiting, the
mammalian NIMA kinase, thereby blocking entry to mitosis
by interacting with phosphorylated regulators such as
Cdc25 and Wee1 (Lu et al., 1996; Yaffe et al., 1997b; Shen
et al., 1998).
Raf1 signaling in Drosophila (Chang and Rubin, 1997). Presumably, interacting with different 14-3-3 isoforms enables
SP and SPAK, as well as other CETS proteins, to perform
functions uniquely suited to specialized physiological responses.
Biological Function of the SP/14-3-3/SPAK Interactions
The actual mode of interactions of SP and its mutant variants with other proteins can be resolved only by structural
studies of each particular case. However, because a high
degree of similarity exists between members of the protein
family, we can try to relate structure to function by examining the relative positions of altered amino acids in the threedimensional protein structures.
In general, default binding could be the result of alterations in the actual protein–protein association domains. Alternatively, it may be secondary to alterations in ligand
binding or to major conformational changes.
The results presented here show that the T65I and G100E
(or D) mutations, but not P74L, alter the pattern of interactions between SP and other proteins. The P74L mutation
does not affect the protein–protein interactions reported in
this study, suggesting that not every mutation that renders
the SP gene inactive also interferes with the physical associations with other proteins. It also implies that the active site,
that is, ligand binding and protein association functions,
may be separated in SP. This is not surprising because
P74L is within the DPDXPD motif and thus expected to disrupt the ligand binding site and not necessarily to interfere
with protein associations. A spatial and functional separation of the protein–protein interaction domain and the catalytic site was recently demonstrated, for example, for the
Ras-SOS complex (Hall et al., 2001).
The mutations that hinder the binding of SP to SIPs,
T65I and G100E, are more distant from the ligand binding
site (see the legend to Figure 7A). They may therefore alter directly and thus identify the protein–protein association domains of SP.
The most reasonable structural inference for such a
role is provided by the G100E (or D) mutation. G100 is situated at the base of a loop structure that protrudes from
the side of the central -sheet. It is likely that exchange of
glycine with the bulky, and potentially charged, glutamate
or aspartate side chains will perturb the vicinity of G100,
which in turn could directly affect protein–protein interactions.
Interpretation of the T65I mutation is more complicated
because it is situated in the top middle section of the main
-sheet, whose integrity is probably very important for obtaining the overall protein structure. Loss of a hydrogen
bond between the threonine 65 hydroxyl group and glutamine 127, the result of the substitution by isoleucine (Figure
7A, dotted line), could primarily destabilize the central -sheet,
Suppression of SPAK, by either antisense or dominant-negative expression, does not alter plant architecture. This is
not surprising for several reasons. Tomato plants have been
cultivated and extensively bred for more then 500 years;
however, despite the readily scored phenotype, only one locus for determinate phenotype was found. Suppression of
gene activity by the antisense or dominant negative approaches is frequently inefficient, and there are at least five
SPAK-like genes with possible redundant function. And because the determinate phenotype is very sensitive to modifiers, it is also possible that the differential response of
meristems and fruits to the inactivation of SPAK is mediated
by as yet unknown organ-specific modifiers.
It is nevertheless satisfying that suppression of SPAK
activity induces elongated fruits, presumably as a result
of effect on cell division. In Aspergillus and in mammalian
cells, the NIMA kinase is required for entry into mitosis
and for the nuclear localization of the cyclin B/cdc2 complex. It may thus participate, redundantly, in regulating
the transition to flowering that is always associated with
an altered distribution of cell divisions in the apical meristem (Bernier, 1988).
The effects of overexpressing 14-3-3s genes, in complementing the loss of the sp function, are more amenable to
interpretation through direct interactions of 14-3-3 with SP
and SPAK, even though indirect effects cannot be excluded.
Although antisense expression had no effect, overexpression of each of the two 14-3-3 genes ameliorated the effects
of sp mutation by increasing the indeterminacy of the shoot
apical meristem, and by increasing the vegetative properties
of the inflorescence in sp an double mutants. These effects
can be accounted for in at least two reasonable ways. It
may be that a given overexpressed 14-3-3 replaces the endogenous legitimate 14-3-3 partner of the mutant sp/14-3-3/
SPAK or that the abundance of 14-3-3 proteins now allows
the replacement of a defective sp protein with a competitive, partially functional, 14-3-3/14-3-3/SPAK association.
Alternatively, if an antagonist of SP, such as FT, functions
via a similar SPAK/14-3-3–based system that involves other
NIMA-like kinases and 14-3-3 homologs, then overexpression of illegitimate 14-3-3 might reduce the efficiency of this
antagonistic system. A dominant-negative effect, via their
involvement in assembling signaling complexes, was also
suggested for 14-3-3s involved redundantly in the Ras1-
T65 and G100 May Identify the Protein Association
Domain Required for SP Function
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The Plant Cell
deforming the surrounding secondary structure, and thus
lead to changes in protein–protein interactions.
Alternatively, a more direct role for the T65 site in the
protein associations of SP may be envisaged. In the SP
model, the threonine side chain lies at the bottom of a surface-accessible hydrophobic depression (Figure 7B) whose
outer rim is lined with polar residues, suggesting that this
site may be directly involved in protein–protein interactions
(Lo Conte et al., 1999). It is very similar to the hydrophobic
pocket that is so critical for the formation of the Ras-SOS
complex mentioned above. By contrast, in both the human
and bovine PEBP structures, the depression is occluded by
loops from Gln-127 to Leu-131 and the top of the adjacent
helix (Lys-157/Tyr-158). In the CEN structure, the loop between residues 130 and 142 is disordered, and it was not included in the structure (Banfield and Brady, 2000); the plant
proteins have significant sequence differences in the disordered area, when compared with the mammalian PEBPs.
This may be an indication of association preferences and
functional differences between plant and mammalian PEBPs.
Conclusions
Figure 7. Molecular Model of the SP Protein.
(A) This molecular model of the SP protein was built using the SwissProt automated comparative protein modeling server, based on its
sequence homology to three members of the PEBP protein family
whose structures have been determined by x-ray crystallographic
methods: human PEBP, bovine brain PEBP, and Antirrhinum CEN
(Protein databank accession numbers 1BEH, 1A44, and 1QOU, respectively). The overall protein model contains extensive stretches
of secondary structure, with a large -sheet (yellow ribbons) in the
center flanked on one side by a smaller -sheet and on the other
side by three short -helices (red tubes). The putative ligand binding
sites, comprised of a number of amino acid residues, which are not
a contiguous sequence, have been delineated in all three existing
crystal structures as a pocket formed between the helices and the
bottom of the central -sheet. The putative active site is indicated by
the position of H86 (purple), surrounded by a black oval. Residues
that affected protein–protein interactions when mutated are T65 (orange) and G100 (blue). Mutation of P74 (red) did not affect protein–
protein interactions. Q127 (cyan) may form a hydrogen bond to the
T65 hydroxyl group.
(B) The molecular surface (transparent gray) superimposed on a
close-up of the SP model (the colors are the same as in [A]) in the vicinity of T65 is shown. This residue lies at the bottom of a depres-
Our studies support the view that to regulate shoot architecture, CETS proteins were recruited to function as a hub of
signaling systems that have the inherent flexibility and potential to integrate a wide variety of developmental cues.
Flexibility and diversity are facilitated by the potential of
CETS proteins to bind diverse classes of regulatory proteins. In mammals, RKIP, a PEBP, was shown to interact
with Raf1 (Yeung et al., 1999, 2000). More recently, Yeung
et al. (2001) also showed that RKIP interacts physically with
two other members of the MAPKKK family, NIK and TAK1,
to modulate the response of NfkB pathway to TNF- and
other signals. Here, we have expanded the range of interacting proteins to include a newly identified plant kinase of
the NIMA class, adapter factors of the 14-3-3 family, a transcription factor of the G-box binding family, and a novel
protein, SIP4. Thus, the diversity of phenotypes regulated
by CETS genes is likely the result of their temporal and spatial association with these and most probably other factors.
We conclude, therefore, that the CETS genes encode a new
family of modulator/adapter proteins analogous to those of
the 14-3-3 family. They seem likely to participate in many
signaling pathways, but their developmental role may be revealed only in systems and organs in which their function is
responsible for a rate-limiting process.
sion that may be important for protein–protein interactions because
mutation of the threonine to isoleucine hinders SP–SIP interactions.
Self-Pruning Interacting Proteins
METHODS
Plant Material
Lycopersicon esculentum lines, VFNT-Cherry SP (LA2756), VFNTCherry/sp2 (LA2705), and anantha seed were provided by R.
Chetelate and C.M. Rick (University of California, Davis). VF36 sp
seed were provided by S. McCormick. Tomato Lycopersicon pennellii lines for mapping (Eshed and Zamir, 1995) were kindly provided by
D. Zamir.
Two-Hybrid Screen
A cDNA library, representing 106 independent EcoRI-XhoI cDNA
clones, was prepared in the Hybrizap vector (Stratagene) from the
mRNA of wild-type apices, 0.5 cm long, containing the second and
third shoot segments. The HF7c yeast line was used as the host because its growth on the histidine (His) selective medium is completely suppressed (Feilotter et al., 1994). Procedures and controls
were as recommended by the manufacturer (Stratagene). The bait
was a full-length SP protein. Full-length SP and sp P74L clones
(Pnueli et al., 1998) were inserted as BamHI blunt/XhoI fragments
into EcoRI blunt/SalI site of the GAL4 binding domain in the pBDGAL4 phagmid vector. SP 1-42 and 43-175 were recovered as EcoRI
and EcoRI/XhoI fragments, respectively, and SP 38-95 was a polymerase chain reaction (PCR) product. All were ligated into a blunt
EcoRI site of the pBD-GAL4 vector.
Full-length EcoRI-XhoI inserts of 14-3-3/2, 14-3-3/74, SIP4, SPAK
270-609, SPAK 389-609, and the partial SPGB were ligated into the
EcoRI-SalI site of the GAL4 BD vector. CEN (a gift of Enrico Coen)
and TFL1 and FT (gifts of Detlef Weigel) were cloned into a bluntended EcoRI site of the bait plasmid.
SPAK 389-609, 270-609, 417-609, and 511-609 were recovered
from the AD clones as above. SPAK 389-443 was an Nde blunt/XhoI
derivative of SPAK 389-609.
SPAK 403-609, 409-609, and 389-609 S406A, described in Figure
4D, were amplified by PCR using their own and the T7 primers and
inserted in the EcoRI blunt site of the bait vector.
2699
Site-Specific Mutagenesis
The T65I and G100E alleles of SP were obtained by a two-step PCR
protocol (Higuchi et al., 1988). The G100D allele of SP was derived
by the same procedure from the G100E clone. Entire sequences
were verified on both strands.
Cloning for in Vitro Association Assays
For in vitro transcription/translation, full-length EcoRI-XhoI inserts of
14-3-3/74, SIP4, and the full-length SPAK cDNA clone were cloned
into pBS SK. To generate GST fusions, full-length SPAK, partial
SPAK 270-609 14-3-3/74, SIP4, and the SIP4 fragments 1-75
(EcoRI-ClaI) and 76-99 (ClaI-XhoI) were cloned as overhang or bluntend fragments in the EcoRI-XhoI site of pGEX 4T-1 (Pharmacia). For
expression in insect Sf9 cells, SP was tagged with the FLAG epitope
by using regular PCR, and the product was cloned into pFastBac
HTb vector in the NcoI/SalI site.
Cytological Procedures
Digoxygenin-labeled (Boehringer Mannheim) RNA probes and hybridization procedures were as referred to in Pnueli et al. (1994) and
Hareven et al. (1996). The tomato RNR2 clone (E. Lifschitz and M.
Egea-Cortines, unpublished data) was subcloned in the same vector,
and the RNA probe was prepared by the same procedure.
In Vitro Binding Assays
35S-methionine–labeled proteins were prepared using the TNT-coupled system (Promega). Binding assays between GST fusion proteins and 35S-methionine–labeled proteins were performed as in
Ausubel et al. (1988). The SP protein was expressed using the Bacto-Bac expression system (Gibco BRL) and immobilized on antiFLAG agarose beads (Sigma). 35S-labeled protein was added that
was supplemented with 10% BSA. The proteins were incubated for
2 hr at room temperature. Binding was performed in 50 mM
K4P2O7, pH 7.5, 150 mM KCl, and 1 mM MgCl2. The samples were
resolved by SDS-PAGE and visualized using a PhosphorImager
(FUJIX BAS 1000, Tokyo, Japan).
Transgenic Plants
SPAK, 14-3-3/74, and 14-3-3/2 cDNAs were cloned, in both sense
and antisense orientations, in pCGN1548 and expressed under the
control of the Cauliflower mosaic virus 35S promoter (Benfey and
Chua, 1990). RK9/8 (Pnueli et al., 1994; Hareven et al., 1996) and
sp;an plants for transformation were maintained in culture vessels by
using cuttings. Leaf disc transformation was conducted essentially
according to McCormick (1991).
Constructs for Transgenic Plants
The full-length 14-3-3/2 and 14-3-3/74, and the full SPAK clone and
the partial SPAK clone 403-609 (Figure 4), were recovered as EcoRIXhoI fragments, cloned into a blunt SalI-SmaI site of a pUC18 between the cauliflower mosaic virus 35S promoter and the nopaline
synthase terminator and subsequently inserted into the pCGN1548
vector as XbaI fragments.
Kinase Assays
Full-length GST:SPAK was expressed at 30C and immobilized,
alone or with a target protein, on glutathione beads. Beads were
washed with phosphorylation buffer (50 mM Tris-HCl, pH 7.5, and 10
mM MgCl2) and incubated in the presence of 2 mM DTT, 2.5 M
ATP, and 2 pM 32P-ATP (3000 Ci/mmol) for 30 min at room temperature. After three washes in PBS buffer, samples were analyzed on
SDS-PAGE or used for binding assays, in which case radioactive
ATP was omitted.
Accession Numbers
The GenBank accession numbers for proteins in this article are as
follows: SPAK, AF079103; SIP4 (I4), AF175963; 14-3-3/2, AF079104;
14-3-3/74, AF079450; and 14-3-3/5, AF079451.
2700
The Plant Cell
ACKNOWLEDGMENTS
The relentless and diligent efforts of David Smyth in preparing the
manuscript are highly appreciated. Thanks are also due to Leo
Brady, Ry Wagner, and Stan Alvarez for their helpful suggestions. We
thank Detlef Weigel for the unconditional gifts of the FT and TFL1
clones and Enrico Coen for the generous gift of the CEN clone. We
thank Dani Zamir for permission to use the tomato insertion lines and
Yona Kasir for her continuous advice on the yeast system. The plant
architecture research program was supported by a grant to E.L. from
the United States–Israel Binational Agricultural Research and Development Fund (BARD), and by additional support from the Israel
Academy of Science, the Israel/USA Bi-national Science Foundation
(BSF) and by Grant No. QLK5-CT-2000-00357 from the European
Commission. Dr. Lilac Pnueli received the Levi Eshkol award from
the Israel Ministry of Science.
Received July 20, 2001; accepted September 17, 2001.
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Tomato SP-Interacting Proteins Define a Conserved Signaling System That Regulates Shoot
Architecture and Flowering
Lilac Pnueli, Tamar Gutfinger, Dana Hareven, Orna Ben-Naim, Neta Ron, Noam Adir and Eliezer
Lifschitz
Plant Cell 2001;13;2687-2702
DOI 10.1105/tpc.010293
This information is current as of June 17, 2017
References
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