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 Lifschitz

a,1

a

Department of Biology,

Science and Technology, Technion, Israel Institute of Technology 32000, Haifa, Israel

b

Department of Chemistry and Institute of Catalysis, Science and Technology, Technion, Israel Institute of Technology 32000, Haifa, Israel

Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and repro-ductive organs from the apical meristem. The

SELF-PRUNING

(

SP

) gene of tomato is a member of a novel

CETS

familyof regulatory genes (

CEN

,

TFL1

, and

FT

) that controls this process. We have identified and describe here several pro-teins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, anovel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 pro-teins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect ofthe

sp

mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational featuresknown to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are re-quired for the biological function of SP and that ligand binding and protein–protein association domains of SP may beseparated. We suggest that

CETS

genes encode a family of modulator proteins with the potential to interact with a va-riety of signaling proteins in a manner analogous to that of 14-3-3 proteins.

INTRODUCTION

The shoot systems of flowering plants display great varia-tion in their architecture and growth habit. The majority ofthis variation can be attributed to modifications of the funda-mental branching pattern (Bell, 1992). These modifications re-sult 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, theapical meristem is indeterminate and active throughout theplant life cycle so that all appendages (leaves, sidebranches, and floral buds) are clearly lateral. A decision toflower is made only once during the life cycle of these spe-cies, after which the main shoot and axillary buds developreproductive organs. This results in a clear distinction be-tween vegetative and reproductive phases.

In contrast, vegetative and reproductive phases alternateregularly along the compound (sympodial) shoots of tomato.The primary vegetative apex is terminated by an inflores-cence after six to 20 leaves have formed, but upwardgrowth then continues from a new vegetative shoot arising

from the uppermost axillary bud just below the terminatinginflorescence. From then on, the stem is composed of reit-erated units, each with three nodal leaves and a terminal in-florescence. Termination of each vegetative apex is thussynonymous with the transition to flowering in tomato butnot in Arabidopsis or Antirrhinum. These basic differences inthe meristematic environments are reflected in the changesincurred by homologous mutations on the overall architec-ture of the two plant species (Pnueli et al., 1998).

Mutations in the

TERMINAL FLOWER1

(

TFL1

) gene of Ar-abidopsis or in the

CENTRORADIALIS

(

CEN

) gene in Antir-rhinum (Shannon and Meeks-Wagner, 1991; Alvarez et al.,1992; Bradley et al., 1996, 1997) convert the inflorescenceapical meristem from a normally indeterminate to a determi-nate state after a few flowers are formed. This results prima-rily in a shorter inflorescence shoot bearing a terminalflower. Yet an altered termination pattern in tomato resultsin an overall dramatic change in the plant architecture. A re-cessive mutation in the

SELF-PRUNING

(

SP

) gene (Yeager,1927; MacArthur, 1932; Pnueli et al., 1998) confers acceler-ated termination of stem units until the shoot is eventuallyterminated by two consecutive inflorescences. Furthermore,in contrast to mutations in the

CEN

and

TFL

genes,

sp

mu-tation is inconsequential for the architecture of the inflores-cence itself, because, unlike that in Arabidopsis, theinflorescence in tomato is inherently determinate (Pnueli etal., 1998). The “determinate” habit of the main shoot of to-mato is repeated in side shoots, resulting in a limited growth

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 atwww.plantcell.org/cgi/doi/10.1105/tpc.010293.

2688 The Plant Cell

of each shoot, a bushy compact constitution, and nearly ho-mogeneous fruit setting. Introduction of the recessive

sp

gene into tomato cultivars revolutionized the tomato indus-try because the determinate growth habit facilitates me-chanical harvesting (Atherton and Harris, 1986). Determinate

sp

plants also display a range of pleiotropic effects: inter-nodes are shorter, more flowers are formed per inflores-cence, control of apical dominance is relaxed, seedgermination is accelerated, and plants are more prone toauxin treatment. In wild-type plants, auxin and its synergistsinduce determinacy and other characteristics of the

sp

phe-notype (Zimmerman and Wilcoxon, 1942; Zimmerman andHitchcock, 1949; Teubner and Wittwer, 1957; Gardner andHedger, 1959; Lifschitz, 1965). In addition, the phenotypicexpression of

sp

varies considerably in different geneticbackgrounds (Pnueli et al., 1998), suggesting to us that thefunction of the

SP

gene is directly, or indirectly, mediated byauxin.

The

CEN

,

TFL

, and

SP

genes have been cloned andshown to be closely related (Bradley et al., 1996, 1997;Pnueli et al., 1998). Thus, although homologous gene muta-tions have different consequences in different shoot sys-tems, in each case it seems that these genes determine thepotential for continuous growth of the shoot apical mer-istem. This is because contrary to the mutant effect, overex-pression 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 genefamily (Kardailsky et al., 1999; Kobayashi et al., 1999). Theproduct 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. Carmel-Goren, personal communication) and six in Arabidopsis (D.Weigel, personal communication), that encodes 23-kD pro-teins. They share sequence similarity with a group of mam-malian polypeptides designated phosphatidylethanolaminebinding proteins (PEBPs; Grandy et al., 1990; Schoentgenand Jolles, 1995). The ability of PEBPs to bind phospholip-ids in vitro promoted the suggestion (Bradley et al., 1997)that they play a role in signaling. The crystal structures ofPEBP from human and bovine sources (Banfield et al., 1998;Serre et al., 1998) and that of the CEN protein from Antirrhi-num (Banfield and Brady, 2000) have been determined.Structural analysis of CEN suggests that its ligand bindingpocket is incapable of accommodating phospholipid andthus is unlikely to function via direct contact with lipid bilay-ers. It is capable of accommodating phosphoryl groups,however, suggesting that PEBP proteins may mediate sig-naling via their association with phosphorylated proteins. In-deed, a mouse PEBP, RKIP, was recently shown to interactwith and inhibit activity of the kinase Raf1 (Yeung et al.,

1999, 2000). Rather than using the inappropriate designa-tion PEBP, we suggest that this gene family be named the

CETS

genes after the first three plant genes with identifiedbiological functions—

CEN

,

TFL1

, and

SP

.

CETS

genes play a critical role in shaping plant architec-ture that is conserved among species.

CETS

genes do not,however, encode proteins that belong to families of DNAbinding proteins, transcription activators, kinases, or recep-tors that are known to regulate major developmental pro-grams 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 vegetativeto reproductive growth.

To understand the molecular function of this new group ofplant regulatory factors, we have initiated the identificationand isolation of SP-interacting proteins (SIPs). It is believedthat the combination of mutant phenotypes, interacting pro-teins, and resolved crystal structures will lead eventually toa comprehensive understanding of the mechanisms that fa-cilitate the reproductive, species-specific architecture offlowering 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 toscreen 10

6

clones of an activation-domain fusion library pre-pared from RNA of shoot apices of wild-type tomato (seeMethods). The scale is not exhaustive, and thus far five pu-

Table 1.

Characteristics of SIPs

Number Type Two Hybrid

a

cDNA

a

Locus

b

SIP2 14-3-3/2 258 258 11-1, 11-2SIP74 14-3-3/74 252 252 4-3SIP3 SPAK

c

339,220 609 2-1, 2-2 (?)SIP4 Novel 98 99 11-1SIP8 SPGB

d

62 ND

e

2-4, 2-5

a

Length (amino acids).

b

Linkage assignments were obtained using 50 tomato lines contain-ing overlapping insertions of the heterologous

Lycopersicon pennellii

wild species (Eshed and Zamir, 1995). SIP2, for example, wasmapped to a region on chromosome 11 shared by insertions 11-1and 11-2. The question mark in the last column indicates that themapping of SIP3 to IL2-2 was not conclusive. Only clones that wereidentified in the primary screen, using SP as a bait, are included inthe table.

c

SPAK, NIMA-like kinase.

d

SPGB, SIP8.

e

ND, not determined.

Self-Pruning Interacting Proteins 2689

tative SIPs were identified. Their basic characteristics areshown in Table 1.

SIP2 and SIP74 (Table 1) represent isoforms of the 14-3-3family. One other interacting member of the 14-3-3 family,14-3-3/5, is not included in Table 1 because it was isolatedin a subsequent, limited, two-hybrid screen using theshorter SIP3 clone (Figrure 1A) as bait, and it has not beengenetically mapped. SIP2 is a novel isoform of 14-3-3, andSIP74 represents the epsilon isoform. Members of this fam-ily are referred to in this article as 14-3-3/2, 14-3-3/74, and14-3-3/5, respectively. 14-3-3s are a class of adapter pro-teins involved in signaling, transcription, and compartmen-talization via their ability to stabilize, dimerize, or bridge theirsubstrates, 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 (forSP-associated kinase). SPAK shares

�

60 to 65% similarity,in its catalytic domain, with the NIMA-like kinases (for neverin mitosis A). Two independent clones extending for differ-ent lengths to the N terminus were isolated (Table 1). A full-length cDNA, encoding 609 amino acids, was isolated sub-sequently from our regular cDNA library. Six related se-quences were identified in the Arabidopsis sequencedatabase. The NIMA kinase regulates early and late pro-gression of G2 stages in

Aspergillus

and mammalian cellsand is required for entry into mitosis and for the nuclear lo-calization 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 sug-gested (Cortez and Elledge, 2000) that the NIMA kinase mayserve as the target for Chfr, a newly identified mitotic check-point protein regulating chromosome condensation (Scolnickand Halazonetis, 2000). SIP4 is a short, 10-kD (99–aminoacid) novel protein. It is related to two anonymous genomicsequences from the Arabidopsis genome project. SIP8,named SPGB, is a putative bZIP transcription factor, a sub-class of G-box (CCACGTGG) binding proteins (Giuliano etal., 1988; Menkens et al., 1995). It has been shown that G-boxfactors are phosphorylated before occupying their position inthe transcription complex (Harter et al., 1994) and that 14-3-3isoforms are components of transcription complexes con-taining G-box binding factors (Lu et al., 1992). On the basis ofthe 62 C-terminal amino acids sequence currently available,the family member most similar to SPGB is GBF4 (Menkensand Cashmore, 1994).

Tomato SIPs Bind to CETS Proteins from Arabidopsis and Antirrhinum

To explore the relevancy of the SP/SIPs associations, wehave investigated whether their interactions are conservedin other species (Figure 1A). The

CEN

gene of Antirrhinumand

TFL1

of Arabidopsis are orthologs of

SP

(Pnueli et al.,

1998) and maintain, in their respective species, the indeter-minate state of the inflorescence meristem (Amaya et al.,1999). The determinate phenotype in tomato is comple-mented by

CEN

(Pnueli et al., 1998), and

SP

, like

CEN

, de-lays flowering in tobacco plants; it also induces a

leafy

-likephenotype in

tfl1

mutant plants of Arabidopsis (results notshown). FT is a functionally divergent CETS protein from Ar-abidopsis that apparently plays a role antagonistic to that of

TFL1

(Kardailsky et al., 1999; Kobayashi et al., 1999). In ad-dition to these three homologs, we have tested three over-lapping peptides of SP spanning residues 1-42, 38-95, and43-175. They are collectively denoted, in the second columnof Figure 1A, by a

�

SP. None of the five SIPs bound to anyof the truncated SP proteins. In contrast, both CEN andTFL1 bind SPAK, 14-3-3/74, and the SPGB proteins but donot react significantly with SIP4. FT, the functional antago-nist of TFL1, displays the same binding pattern as TFL1.Thus, some of the SIP interactions in tomato are apparentlyconserved in distantly related plants. The interactions of FTwith SIPs, however, do not provide a molecular basis for itsantagonistic role in flowering.

SIPs Form a Network of Bipartite Interactions

The molecular identity of the SIPs supports their involve-ment in signaling processes. To test the possibility that theyare involved in a SP-dependent signaling network, we ex-amined them for their ability to form associations amongthemselves. Three 14-3-3s were included in some of thepairwise tests to probe possible differential affinities with theother SIPs or among themselves. Results of the two-hybridtests are shown in Figures 1B and 1C (and supporting invitro assays are discussed in relation to experiments docu-mented 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 (Figure1B). SP, by contrast, does not form homodimers (data notshown), which is consistent with the conclusion derivedfrom the structural analysis of CEN (Banfield and Brady,2000). Evidently, 14-3-3 isoforms, initially recovered as in-teracting with SP, also interact with SPAK and SIP4. Be-cause G-box binding proteins are known to interact with 14-3-3s, the lack of interactions between 14-3-3s and theSPGB is attributed to the short fragment of the gene avail-able to us. Presumably, SP and 14-3-3s are associated withseparate domains of the GB protein.

Corroborating

�

-galactosidase assays are documented inFigure 1C. Interactions involving SPGB were omitted from thistest because they were negative in the much more sensitiveassay shown in Figure 1B. Strong interactions occur be-tween SPAK and 14-3-3/74, and between SIP4 and 14-3-3/74. SPAK and SIP4 show weaker, albeit differential, interac-tions with 14-3-3/5 and 14-3-3/2. Interestingly, 14-3-3/74,which weakly dimerizes, forms a very strong association

2690 The Plant Cell

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 modi-fications 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, byin situ hybridization, with the expression pattern of

SP

.

SPAK

is expressed from the beginning in the apical mer-istem and in the leaf and stem vasculature of the primaryapex of a young, two-leaf seedling (Figure 2A). A similar pat-tern is observed in the floral primordia of the primary inflo-rescence (Figure 2B) and in the vegetative meristem of thefirst sympodial segment (Figure 2B). In the developing floralbud,

SPAK

expression is particularly prominent in the sta-mens and carpels (Figure 2C). This expression pattern of

SPAK

overlaps spatially and temporarily with the patternsobserved for

SP

and for the tomato

LEAFY

gene (Pnueli etal., 1998).

The in situ localization of transcripts of the

SPGB

and

SIP4

genes is shown only for the apical meristems of youngseedling (Figures 2D and 2E) because they too overlapthroughout with that of

SPAK

and

SP.

In addition, the samepattern was seen for 14-3-3/2 (data not shown). Because allthree probes shown in Figures 2A to 2E, along with

SP

andthe

T-Leafy

probes, mark the same meristematic domains,we have used the tomato ribonucleotide-reductase smallsubunit gene (

RNR2

; E. Lifschitz and M. Egea-Cortines, un-published data) as a positive control probe. This gene is ex-pected to be expressed at high levels in meristematictissues 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 theSIPs to associate with SP and among themselves, indepen-dent of potential yeast partners. A FLAG-SP fusion protein,expressed in Sf9 insect cells, was tested in pairwise combi-nations with radiolabeled 14-3-3/74, SPAK, and SIP4 pre-

Figure 1.

Identification and Characterization of the SIPs in the YeastTwo-Hybrid System.

(A)

Conserved interactions between SIPs and SP homologs from Ar-abidopsis and Antirrhinum

.

Results of the two-hybrid tests betweenCETS and SIPs are illustrated. The FT column was inserted from aseparate plate because BD-FT displays a residual growth (meaningactivation potential) on the

�

His selective medium, thus requiringtests in plates supplemented with 4 mM triamino triazole (3AT).

�

SPrefers collectively to three overlapping peptides of SP (see text), allof 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, two-hybrid test, on the

�

His selective medium, are shown.

(C)

Colony lift,

�

-galactosidase association assays between SIPsare shown.The strength of the interactions ranges from 11 Miller units for theSPAK:14-3-3/74 pair to 0.4 units for SPAK:14-3-3/2. Note that forthe presence of positive interactions, a positive control between p53and SV40 is not included here; P53 is used as a negative control.

Self-Pruning Interacting Proteins 2691

pared in the in vitro transcription/translation system and, asshown in Figure 3A, sections I to III, interacts readily with allthree tested proteins. Identical results were obtained withFLAG-SP expressed in yeast cells (results not shown). Invitro binding assays shown in Figure 3A, section IV, verifiedalso the in vitro association between SPAK and 14-3-3/74,the lack of association between SPAK and SIP4, and thepotential of SPAK to dimerize. The results of the in vitrostudies thus are consistent with the results of the two-hybridexperiments illustrated in Figure 1.

SPAK and 14-3-3/74 Interact in aPhosphorylation-Dependent Manner

For the in vitro reciprocal binding assays between pairs ofSIPs, we used GST fusion proteins expressed in bacteriaand

35

S-methionine–labeled proteins translated in vitro. Inpreliminary experiments, some pairwise reciprocal assays

did not give consistent results. It was subsequently deter-mined that SPAK and SIP4 (used, for example, in the exper-iments described in Figure 3A), but not 14-3-3/74, areactually phosphorylated by kinases present in the in vitrotranslation system.

Therefore, for the initial evaluation of the role of phosphor-ylation in interactions between SIP proteins, the possibilitythat SPAK is autophosphorylated, and that it phosphory-lates other SIP proteins, was investigated. For autophos-phorylation, we tested the ability of full-length SPAK tophosphorylate a truncated protein deleted for its catalyticdomain (amino acids 1 to 270; see Figures 4A to 4C). Asshown in Figure 3B, secton I, lanes 1 and 2, the deletedSPAK protein is indeed phosphorylated by full-length SPAKexpressed in bacteria. Of the other SIPs, only SIP4 wasphosphorylated, in vitro, by SPAK (Figure 3B, section I, lane3), thus establishing a biochemical function for SPAK. Dele-tion analysis of SIP4 suggested that the phosphorylationsite was present in the C-terminal 20 amino acids (Figure

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 sub-unit 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 polymer-ase. C, carpel; FP, floral primordium; L, leaf; P, petal; PR, primary apex; S, sepal; SA, sympodial apex; ST, stamen; VS, vascular strand.

2692 The Plant Cell

3B, section I, lanes 4 and 5), where the only candidate is aserine 98, embedded in a potential phosphorylation domain(R/KXXS/T).

Because in many cases 14-3-3 binds phosphorylatedproteins (Yaffe et al., 1997b; Finnie et al., 1999), the require-ment for SPAK and SIP to be phosphorylated to associatewith 14-3-3/74 was examined. Pull-down assays shown inFigure 3B, sections II and III, revealed that in vitro–translated14-3-3/74, which by itself is not phosphorylated, binds onlyphosphorylated SPAK (Figure 3B, section II); however, toform homodimers, 17-3-3/74 needs not to be phosphory-lated. Likewise, it was found that only a phosphorylatedSIP4 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 toboth SP and 14-3-3 proteins. Another serine/threonine ki-nase, Raf1, provides a precedent because it is associatedwith 14-3-3 (Rommel et al., 1996; Muslin et al., 1996; Yaffeet al., 1997a; Rittinger et al., 1999) and with a mammalianhomolog of SP, RKIP (Yeung et al., 1999, 2000). The inter-actions of 14-3-3 and SP with SPAK were therefore studiedin more detail. The amino acid sequence of SPAK is shownin Figure 4A. The deduced catalytic and dimerization do-mains, and the binding sites for SP and 14-3-3/74, areshown schematically in Figure 4B. Deletion analysis in theyeast two-hybrid system suggests that the C-terminal do-

Figure 3. SP and SIPs Interact in Vitro.

(A) In I to III, in vitro association of labeled 14-3-3/74, SPAK, andSIP4, respectively, to FLAG-SP protein is shown. The FLAG-SP genewas expressed in Sf9 cells. Proteins from expressing cells and control

cells were immobilized on anti-FLAG agarose beads, incubated with35S-Met–labeled SIPs, resolved on SDS-PAGE, and visualized by au-toradiography. In IV, SPAK polypeptides form homodimers and in-teract in vitro with 14-3-3/74 but not with SIP4. Note that SPAK inIV, as well as SPAK and SIP4 in II and III, respectively, are phos-phorylated.(B) Phosphorylation-dependent associations between SIP proteins.In I, SPAK auto-phosphorylates and also phosphorylates the C-ter-minal region of SIP4. A GST fusion with the complete SPAK proteinwas incubated in a phosphorylation reaction assay with a GST con-trol (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); anda GST-SIP4 1-75 N-terminal domain (lane 5). Proteins were sepa-rated by SDS-PAGE and autoradiographed. In II, 14-3-3/74 dimerizesand interacts with phosphorylated (*), but not with nonphosphory-lated, GST-SPAK fusion protein. In III, 14-3-3/74 protein binds toGST-SIP4 only if the latter is phosphorylated. In both II and III, invitro–translated 35S-Met–labeled SIPs (indicated to the right of eachgel) were incubated with immobilized GST-SIP fusion proteins (indi-cated above each gel). The asterisks indicate a GST-SIP previouslyphosphorylated by SPAK. Samples of bound radioactive proteinswere eluted, resolved on SDS-PAGE, and visualized by autoradiog-raphy.

Self-Pruning Interacting Proteins 2693

main of SPAK, amino acids 511 to 609, is required for ho-modimerization (Figure 4C). A binding site for SP must alsolie within the larger 389 to 609 region because it alone waspresent in the smaller of the two original cDNA products thatbound SP (Table 1). A deletion analysis of SPAK also revealsthat a 29–amino acids long peptide, spanning positions 389to 417, inclusive, is required for the binding of both SP and14-3-3/74, but binding is effective only in conjunction with aC-terminal dimerization domain (Figure 4C).

The sequence of the putative, 29–amino acid long, bind-ing domain for SP and 14-3-3/74 contains a classical con-sensus sequence for the binding of 14-3-3 proteins (Yaffe etal., 1997a; Figures 4A and 4D), raising the unexpected pos-sibility that SP and 14-3-3 share the same binding sitewithin SPAK.

To address this possibility, we further dissected the 389-to-417 peptide of SPAK 389-609 into three subregions (Fig-ure 4D) and tested the mutated SPAK proteins against SPand 14-3-3/74 in the yeast two-hybrid system. The resultsnarrowed the essential SPAK site to the six–amino acids se-quence (shown in red) that forms the consensus for thebinding of 14-3-3 proteins. Included in this peptide is a con-servative serine residue in position 406 that is likely to becrucial for effective binding with 14-3-3/74 (Yaffe et al.,1997b; Rittinger et al., 1999). Indeed, replacement of serine406 by alanine (Figure 4D, lines III) abolished the binding ofSPAK 389-609 to 14-3-3/74 and to SP but not the formationof SPAK dimmers, thus providing further evidence that SPand 14-3-3/74 may be interchangeable at, or even competefor, this SPAK site under certain physiological conditions.

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 isindicated by an asterisk.(B) Schematic representation of the major functional domains in the SPAK protein. The scheme is based on data derived from sequence com-parisons 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 interactionwith 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 deletionsof 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 two-hybrid 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 ef-fective 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 re-gion (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 arehighlighted in red. Delineated below are the sequences of the three mutant versions of SPAK 389-417 that were used for the detailed mappingof the common binding site. The first retained the consensus six–amino acid sequence at its N terminus (I), the second polypeptide had this se-quence deleted (II), and the third included the 14-3-3 consensus sequence but with serine 406 replaced by alanine (III). Results of two-hybridtests of interactions between these modified SPAK proteins with SP, SPAK, and 14-3-3/74 are shown at bottom. Serine 406 is required for thebinding of both SP and 14-3-3 under these conditions (plate at the bottom right).

2694 The Plant Cell

In Raf1, two 14-3-3 molecules form a bivalent intramolec-ular 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). Likewisethere is, in addition to the serine 406 site, another potentialbinding site for 14-3-3, RRNS274LP, in the N-terminal do-main of SPAK. To examine the binding potential of this sec-ond site to 14-3-3/74, a full-length SPAK that includes theS274 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 sitefor SP but, like Raf1, at least two potential binding sites for14-3-3.

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 contextof plant architecture, we have initiated their study in trans-genic plants. At first, the 14-3-3 and SPAK genes were usedto transform plants of the determinate double mutant sp an(anantha; Paddock and Alexander, 1952) genotype becauseanantha plants are more sensitive to the growth-promotingeffects of the SP gene in two ways (Pnueli et al., 1998). First,the determinate habit of the sp;an shoots (Figure 5B; cf. Fig-ure 5A) is converted by the overexpression of SP to a highlyirregular, indeterminate pattern, with three to six internodesbetween inflorescences (Figure 5C). Second, the primordiaof the sp;an inflorescence, which are normally arrested at apre-floral, cauliflower-like state (Figure 5E), now developleaflets and leaves (Figures 5C and 5F). Thus, transgenes af-fecting the SP system are expected foremost to alter the de-terminate habit of sp;an shoots and/or the stage in whichthe primordia of the anantha inflorescence are arrested.

No developmental changes were observed in sp or sp;anplants expressing antisense RNA of the 14-3-3/2 or 14-3-3/74 genes. However, a clear manifestation of increased veg-etative characteristics is evident in the shoots and inflores-cence of sp;an plants overexpressing either of the twogenes (Figures 5D, 5G, and 5H). A significant attenuation ofthe determinate phenotype was observed in sp;an plantsoverexpressing 14-3-3/2 (Figure 5D), although this was notas complete as observed when SP is overexpressed in thesame background (Figure 5C). The now indeterminate mainand side shoots of sp;an plants expressing 14-3-3/2 featurea variable number of one to three internodes between inflo-rescences, with spacings of one or two leaves predominat-ing. In addition, the inflorescence is now mildly leafy (Figure5G). Overexpression of 14-3-3/74 in sp;an plants resulted inonly mild, albeit consistent, delay of determinacy, so thatmany branches generated five to seven inflorescenceshoots before termination, as compared with three to fourinflorescence shoots in the progenitor line. However, thesecond 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 compen-sate for the loss of function of the SP gene, at least in theanantha mutant background, suggesting perhaps someoverlapping biochemical functions. It should also be notedthat the partial suppression of the determinate sp mutantphenotype that is a semi-determinate habit is also charac-teristic of several genetic backgrounds such as that of theVF36 line. Modifiers may include variants of SP-interactinggenes such as 14-3-3.

It will be interesting to see if overexpression, in the sameplants, of 14-3-3/2 and 14-3-3/74, which form strong het-erodimers (Figure 1), will result in a more complete comple-mentation of the determinate phenotype.

Antisense expression of SPAK has no obvious effect ongrowth habit. However, all sp plants expressing antisenseRNA form pear-shaped fruits instead of the normal roundfruits of the progenitor plants. Pear-shaped, ovate fruits areformed in transgenic plants bearing regular-size fruits aswell as in plants bearing cherry fruits (Figures 5I and 5J). Ex-actly the same results were obtained when a dominant-neg-ative form of SPAK (i.e., amino acids 403 to 609) wasexpressed in tomato plants. Outcrossing of antisense-expressing plants with different determinate and indetermi-nate lines showed that formation of ovate fruits is indepen-dent 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 withSIPs, we took advantage of the recently determined crystalstructures of three CETS (PEBP) proteins, from mammalsand plants (Banfield et al., 1998; Serre et al., 1998; Banfieldand Brady, 2000), and of single amino acid alterationsknown to inactivate plant CETS genes (Bradley et al., 1996,1997; Pnueli et al., 1998; Kardailsky et al., 1999; Kobayashiet al., 1999). In principle, it should be possible, under thesecircumstances, to relate binding potential of mutant SP vari-ants to conformational features, known to be conserved, inthis new family of regulatory factors.

For a rational choice of mutant sites to be tested, we havebuilt a structural model of SP, using homology-based mod-eling (Peitsch, 1996). The resulting three-dimensional model(see Figure 7A) is very similar to the structures reported forPEBP proteins. We have subsequently analyzed the bindingproperties of SP proteins carrying three known mutations inparticularly critical and conservative domains of all CETS/PEBP proteins.

In the recessive sp allele, proline in position 76 (Pnueli etal., 1998), designated here as P74 to conform with the align-ment of all the genes in the family (Banfield and Brady,2000), is replaced by leucine. Proline 74 is within an invari-ant motif, DPDXP74D, near the putative active site (indi-cated 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 ar-rows), which is the consequence of the sp mutation. The meristems in each inflorescence (arrows) proliferate, and all are arrested in a cauli-flower-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 genera-tion 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 pat-tern 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 primor-dia 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 fromplants expressing p35S::SPAK antisense RNA. (J) Round cherry tomato fruits (left) and elongated cherry fruits (right) from plants expressing adominant-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 mutantalleles of SP are known, we have exploited two other con-served mutant sites of its homolog, the TFL1 gene of Arabi-dopsis (Bradley et al., 1997). One disfunctional allele of TFL1(i.e., a terminal flower and early flowering) carries a threo-nine-to-isoleucine alteration in position 65 and the other aglycine-to-aspartic acid in position 100 (Bradley et al.,1997). These two sites are more distant from the ligandbinding site, and it appears that neither mutation potentiallyalters SP ligand binding.

To investigate the effect of these mutations on the bindingproperties of SP, we introduced identical mutations into theSP gene. A change in one base was sufficient for the T65Ialteration, but two steps were required (G to E and E to D) tointroduce the G100D change in the SP. The three mutantforms of SP were subsequently tested in yeast for their in-teraction with the SIP proteins. As shown in Figure 6, thefour SIPs interact readily with the SP-P74L mutant protein.In contrast, with the exception of the successful binding be-tween SP T65I and the short peptide representing the SPGBprotein, the SP-T65I, SP-G100E, and the SP-G100D mu-tants, as well as the double mutant SP-T65I G100E, abol-ished the SP–SIPs interactions in the two-hybrid test.

DISCUSSION

SPAK, SPGB, and 14-3-3s belong to protein families knownto be involved in signaling pathways; the novel SIP4 may be

phosphorylated by SPAK; and SP and the other SIPs all in-teract with 14-3-3s. The observed network of specific inter-actions between SIPs provides support for the assertionthat the SIPs are legitimate components of a putative SP-dependent signaling system. Upstream effectors and down-stream targets for the SP system are yet to be discovered,and the full range of the biochemical diversification of CETSproteins has not yet been explored. Even so, it is alreadypossible at this point to discuss the possible biochemicalfunction of SP in conjunction with mutations that interferewith its binding properties and in relation to its interactionswith 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 far-reaching implications, with the Raf1 complex. Becausenone of the known SP partners seems likely to anchor thesystem to the membrane, the SP signaling system, like thatof Raf1, is more likely to be a transient component of mem-brane-associated effector complexes. Although Raf1 andSPAK belong to two different families of kinases, they eachbind to members of the same protein family: SP, TFL1, andCEN bind to SPAK, and RKIP, an SP homolog protein frommammals, binds to Raf1 (Yeung et al., 1999, 2000). Both SPand RKIP bind only phosphorylated kinases, the two ki-nases bind 14-3-3s in a phosphorylation-dependent man-ner, and each has two 14-3-3 binding sites. In fact, wefound that the NIMA kinase itself also contains two potentialbinding sites for 14-3-3 proteins. In this context, it is note-worthy that if Raf1, like SPAK, also uses the 14-3-3 bindingsite to attract the mammalian SP homolog RKIP, the anal-ogy is extended and an unexpected additional regulatorypoint for Raf1 is envisaged.

Here, we report that binding of SP to SPAK depends on aserine residue at position 406. The phosphorylation statusof this particular serine has not been directly established,but because the very same site is also required for the bind-ing of 14-3-3/74, and because the requirement for a phos-phorylated serine in the 14-3-3 class of binding sites hasalready been established (Muslin et al., 1996), it is likely thatserine 406 of SPAK must be phosphorylated for SPAK tobind SP as well. It is not implied, however, that the phos-phoryl-serine 406 is actually accommodated by the ligandbinding pocket of SP.

In Raf1, two 14-3-3 molecules form bivalent intramolecu-lar bridging sites in the N- and C-terminal domains beforebeing displaced from the N-terminal site by an activatedRas (Li et al., 1995; Rommel et al., 1996; Muslin et al., 1996).Likewise there are two binding sites for 14-3-3 in SPAK, butit seems as if only one is shared by SP. Exclusive SPbridges in SPAK are currently excluded also because SPdoes not dimerize. However, potentially SP could participatein an intramolecular bridge in cooperation with a 14-3-3 if

Figure 6. Binding Properties of T65I and G100D Mark Putative Con-servative Protein–Protein Association Domains of SP.

SP and its mutant derivatives used in this experiment were taggedwith FLAG (to verify expression in yeast) and fused to the Gal4 bind-ing domain. The SP bait (double, in the far right column) denotes thedouble mutant SP T65I:G100E.

Self-Pruning Interacting Proteins 2697

transient modifications of SPAK by other accessory proteinsoccur. Pin1, a peptidyl-prolyl isomerase, is one possibleagent for such a structural modification of SPAK. Pin1 wasinitially identified as interacting with, and inhibiting, themammalian NIMA kinase, thereby blocking entry to mitosisby interacting with phosphorylated regulators such asCdc25 and Wee1 (Lu et al., 1996; Yaffe et al., 1997b; Shenet al., 1998).

Biological Function of the SP/14-3-3/SPAK Interactions

Suppression of SPAK, by either antisense or dominant-neg-ative expression, does not alter plant architecture. This isnot surprising for several reasons. Tomato plants have beencultivated and extensively bred for more then 500 years;however, despite the readily scored phenotype, only one lo-cus for determinate phenotype was found. Suppression ofgene activity by the antisense or dominant negative ap-proaches is frequently inefficient, and there are at least fiveSPAK-like genes with possible redundant function. And be-cause the determinate phenotype is very sensitive to modifi-ers, it is also possible that the differential response ofmeristems and fruits to the inactivation of SPAK is mediatedby as yet unknown organ-specific modifiers.

It is nevertheless satisfying that suppression of SPAKactivity induces elongated fruits, presumably as a resultof effect on cell division. In Aspergillus and in mammaliancells, the NIMA kinase is required for entry into mitosisand for the nuclear localization of the cyclin B/cdc2 com-plex. It may thus participate, redundantly, in regulatingthe transition to flowering that is always associated withan altered distribution of cell divisions in the apical mer-istem (Bernier, 1988).

The effects of overexpressing 14-3-3s genes, in comple-menting the loss of the sp function, are more amenable tointerpretation through direct interactions of 14-3-3 with SPand SPAK, even though indirect effects cannot be excluded.Although antisense expression had no effect, overexpres-sion of each of the two 14-3-3 genes ameliorated the effectsof sp mutation by increasing the indeterminacy of the shootapical meristem, and by increasing the vegetative propertiesof the inflorescence in sp an double mutants. These effectscan be accounted for in at least two reasonable ways. Itmay be that a given overexpressed 14-3-3 replaces the en-dogenous legitimate 14-3-3 partner of the mutant sp/14-3-3/SPAK or that the abundance of 14-3-3 proteins now allowsthe replacement of a defective sp protein with a competi-tive, partially functional, 14-3-3/14-3-3/SPAK association.Alternatively, if an antagonist of SP, such as FT, functionsvia a similar SPAK/14-3-3–based system that involves otherNIMA-like kinases and 14-3-3 homologs, then overexpres-sion of illegitimate 14-3-3 might reduce the efficiency of thisantagonistic system. A dominant-negative effect, via theirinvolvement in assembling signaling complexes, was alsosuggested for 14-3-3s involved redundantly in the Ras1-

Raf1 signaling in Drosophila (Chang and Rubin, 1997). Pre-sumably, interacting with different 14-3-3 isoforms enablesSP and SPAK, as well as other CETS proteins, to performfunctions uniquely suited to specialized physiological re-sponses.

T65 and G100 May Identify the Protein Association Domain Required for SP Function

The actual mode of interactions of SP and its mutant vari-ants with other proteins can be resolved only by structuralstudies of each particular case. However, because a highdegree of similarity exists between members of the proteinfamily, we can try to relate structure to function by examin-ing the relative positions of altered amino acids in the three-dimensional protein structures.

In general, default binding could be the result of alter-ations in the actual protein–protein association domains. Al-ternatively, it may be secondary to alterations in ligandbinding 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 interac-tions between SP and other proteins. The P74L mutationdoes not affect the protein–protein interactions reported inthis study, suggesting that not every mutation that rendersthe SP gene inactive also interferes with the physical associ-ations 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 becauseP74L is within the DPDXPD motif and thus expected to dis-rupt the ligand binding site and not necessarily to interferewith protein associations. A spatial and functional separa-tion of the protein–protein interaction domain and the cata-lytic site was recently demonstrated, for example, for theRas-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 bindingsite (see the legend to Figure 7A). They may therefore al-ter directly and thus identify the protein–protein associa-tion domains of SP.

The most reasonable structural inference for such arole is provided by the G100E (or D) mutation. G100 is sit-uated at the base of a loop structure that protrudes fromthe side of the central �-sheet. It is likely that exchange ofglycine with the bulky, and potentially charged, glutamateor aspartate side chains will perturb the vicinity of G100,which in turn could directly affect protein–protein interac-tions.

Interpretation of the T65I mutation is more complicatedbecause it is situated in the top middle section of the main�-sheet, whose integrity is probably very important for ob-taining the overall protein structure. Loss of a hydrogenbond between the threonine 65 hydroxyl group and gluta-mine 127, the result of the substitution by isoleucine (Figure7A, dotted line), could primarily destabilize the central �-sheet,

2698 The Plant Cell

deforming the surrounding secondary structure, and thuslead to changes in protein–protein interactions.

Alternatively, a more direct role for the T65 site in theprotein associations of SP may be envisaged. In the SPmodel, the threonine side chain lies at the bottom of a sur-face-accessible hydrophobic depression (Figure 7B) whoseouter rim is lined with polar residues, suggesting that thissite may be directly involved in protein–protein interactions(Lo Conte et al., 1999). It is very similar to the hydrophobicpocket that is so critical for the formation of the Ras-SOScomplex mentioned above. By contrast, in both the humanand bovine PEBP structures, the depression is occluded byloops from Gln-127 to Leu-131 and the top of the adjacenthelix (Lys-157/Tyr-158). In the CEN structure, the loop be-tween residues 130 and 142 is disordered, and it was not in-cluded in the structure (Banfield and Brady, 2000); the plantproteins have significant sequence differences in the disor-dered area, when compared with the mammalian PEBPs.This may be an indication of association preferences andfunctional differences between plant and mammalian PEBPs.

Conclusions

Our studies support the view that to regulate shoot architec-ture, CETS proteins were recruited to function as a hub ofsignaling systems that have the inherent flexibility and po-tential to integrate a wide variety of developmental cues.Flexibility and diversity are facilitated by the potential ofCETS proteins to bind diverse classes of regulatory pro-teins. In mammals, RKIP, a PEBP, was shown to interactwith Raf1 (Yeung et al., 1999, 2000). More recently, Yeunget al. (2001) also showed that RKIP interacts physically withtwo other members of the MAPKKK family, NIK and TAK1,to modulate the response of NfkB pathway to TNF-� andother signals. Here, we have expanded the range of inter-acting proteins to include a newly identified plant kinase ofthe NIMA class, adapter factors of the 14-3-3 family, a tran-scription factor of the G-box binding family, and a novelprotein, SIP4. Thus, the diversity of phenotypes regulatedby CETS genes is likely the result of their temporal and spa-tial association with these and most probably other factors.We conclude, therefore, that the CETS genes encode a newfamily of modulator/adapter proteins analogous to those ofthe 14-3-3 family. They seem likely to participate in manysignaling pathways, but their developmental role may be re-vealed only in systems and organs in which their function isresponsible for a rate-limiting process.

Figure 7. Molecular Model of the SP Protein.

(A) This molecular model of the SP protein was built using the Swiss-Prot automated comparative protein modeling server, based on itssequence homology to three members of the PEBP protein familywhose structures have been determined by x-ray crystallographicmethods: human PEBP, bovine brain PEBP, and Antirrhinum CEN(Protein databank accession numbers 1BEH, 1A44, and 1QOU, re-spectively). The overall protein model contains extensive stretchesof secondary structure, with a large �-sheet (yellow ribbons) in thecenter flanked on one side by a smaller �-sheet and on the otherside by three short �-helices (red tubes). The putative ligand bindingsites, comprised of a number of amino acid residues, which are nota contiguous sequence, have been delineated in all three existingcrystal structures as a pocket formed between the helices and thebottom of the central �-sheet. The putative active site is indicated bythe position of H86 (purple), surrounded by a black oval. Residuesthat affected protein–protein interactions when mutated are T65 (or-ange) and G100 (blue). Mutation of P74 (red) did not affect protein–protein interactions. Q127 (cyan) may form a hydrogen bond to theT65 hydroxyl group.(B) The molecular surface (transparent gray) superimposed on aclose-up of the SP model (the colors are the same as in [A]) in the vi-cinity of T65 is shown. This residue lies at the bottom of a depres-

sion that may be important for protein–protein interactions becausemutation of the threonine to isoleucine hinders SP–SIP interactions.

Self-Pruning Interacting Proteins 2699

METHODS

Plant Material

Lycopersicon esculentum lines, VFNT-Cherry SP (LA2756), VFNT-Cherry/sp2 (LA2705), and anantha seed were provided by R.Chetelate and C.M. Rick (University of California, Davis). VF36 spseed were provided by S. McCormick. Tomato Lycopersicon pennel-lii lines for mapping (Eshed and Zamir, 1995) were kindly provided byD. Zamir.

Two-Hybrid Screen

A cDNA library, representing 106 independent EcoRI-XhoI cDNAclones, was prepared in the Hybrizap vector (Stratagene) from themRNA of wild-type apices, �0.5 cm long, containing the second andthird shoot segments. The HF7c yeast line was used as the host be-cause its growth on the histidine (�His) selective medium is com-pletely suppressed (Feilotter et al., 1994). Procedures and controlswere as recommended by the manufacturer (Stratagene). The baitwas a full-length SP protein. Full-length SP and sp P74L clones(Pnueli et al., 1998) were inserted as BamHI blunt/XhoI fragmentsinto EcoRI blunt/SalI site of the GAL4 binding domain in the pBD-GAL4 phagmid vector. SP 1-42 and 43-175 were recovered as EcoRIand EcoRI/XhoI fragments, respectively, and SP 38-95 was a poly-merase chain reaction (PCR) product. All were ligated into a bluntEcoRI site of the pBD-GAL4 vector.

Full-length EcoRI-XhoI inserts of 14-3-3/2, 14-3-3/74, SIP4, SPAK270-609, SPAK 389-609, and the partial SPGB were ligated into theEcoRI-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 blunt-ended EcoRI site of the bait plasmid.

SPAK 389-609, 270-609, 417-609, and 511-609 were recoveredfrom the AD clones as above. SPAK 389-443 was an Nde blunt/XhoIderivative of SPAK 389-609.

SPAK 403-609, 409-609, and 389-609 S406A, described in Figure4D, were amplified by PCR using their own and the T7 primers andinserted in the EcoRI blunt site of the bait vector.

Transgenic Plants

SPAK, 14-3-3/74, and 14-3-3/2 cDNAs were cloned, in both senseand antisense orientations, in pCGN1548 and expressed under thecontrol of the Cauliflower mosaic virus 35S promoter (Benfey andChua, 1990). RK9/8 (Pnueli et al., 1994; Hareven et al., 1996) andsp;an plants for transformation were maintained in culture vessels byusing cuttings. Leaf disc transformation was conducted essentiallyaccording to McCormick (1991).

Constructs for Transgenic Plants

The full-length 14-3-3/2 and 14-3-3/74, and the full SPAK clone andthe partial SPAK clone 403-609 (Figure 4), were recovered as EcoRI-XhoI fragments, cloned into a blunt SalI-SmaI site of a pUC18 be-tween the cauliflower mosaic virus 35S promoter and the nopalinesynthase terminator and subsequently inserted into the pCGN1548vector as XbaI fragments.

Site-Specific Mutagenesis

The T65I and G100E alleles of SP were obtained by a two-step PCRprotocol (Higuchi et al., 1988). The G100D allele of SP was derivedby the same procedure from the G100E clone. Entire sequenceswere verified on both strands.

Cloning for in Vitro Association Assays

For in vitro transcription/translation, full-length EcoRI-XhoI inserts of14-3-3/74, SIP4, and the full-length SPAK cDNA clone were clonedinto pBS SK. To generate GST fusions, full-length SPAK, partialSPAK 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 blunt-end fragments in the EcoRI-XhoI site of pGEX 4T-1 (Pharmacia). Forexpression in insect Sf9 cells, SP was tagged with the FLAG epitopeby using regular PCR, and the product was cloned into pFastBacHTb vector in the NcoI/SalI site.

Cytological Procedures

Digoxygenin-labeled (Boehringer Mannheim) RNA probes and hy-bridization procedures were as referred to in Pnueli et al. (1994) andHareven 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-cou-pled system (Promega). Binding assays between GST fusion pro-teins and 35S-methionine–labeled proteins were performed as inAusubel et al. (1988). The SP protein was expressed using the Bac-to-Bac expression system (Gibco BRL) and immobilized on anti-FLAG agarose beads (Sigma). 35S-labeled protein was added thatwas supplemented with 10% BSA. The proteins were incubated for2 hr at room temperature. Binding was performed in 50 mMK4P2O7, pH 7.5, 150 mM KCl, and 1 mM MgCl2. The samples wereresolved by SDS-PAGE and visualized using a PhosphorImager(FUJIX BAS 1000, Tokyo, Japan).

Kinase Assays

Full-length GST:SPAK was expressed at 30�C and immobilized,alone or with a target protein, on glutathione beads. Beads werewashed with phosphorylation buffer (50 mM Tris-HCl, pH 7.5, and 10mM MgCl2) and incubated in the presence of 2 mM DTT, 2.5 �MATP, and 2 pM 32P-ATP (3000 Ci/mmol) for 30 min at room tempera-ture. After three washes in PBS buffer, samples were analyzed onSDS-PAGE or used for binding assays, in which case radioactiveATP was omitted.

Accession Numbers

The GenBank accession numbers for proteins in this article are asfollows: 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 themanuscript are highly appreciated. Thanks are also due to LeoBrady, Ry Wagner, and Stan Alvarez for their helpful suggestions. Wethank Detlef Weigel for the unconditional gifts of the FT and TFL1clones and Enrico Coen for the generous gift of the CEN clone. Wethank Dani Zamir for permission to use the tomato insertion lines andYona Kasir for her continuous advice on the yeast system. The plantarchitecture research program was supported by a grant to E.L. fromthe United States–Israel Binational Agricultural Research and Devel-opment Fund (BARD), and by additional support from the IsraelAcademy of Science, the Israel/USA Bi-national Science Foundation(BSF) and by Grant No. QLK5-CT-2000-00357 from the EuropeanCommission. Dr. Lilac Pnueli received the Levi Eshkol award fromthe Israel Ministry of Science.

Received July 20, 2001; accepted September 17, 2001.

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DOI 10.1105/tpc.010293 2001;13;2687-2702Plant CellLifschitz

Lilac Pnueli, Tamar Gutfinger, Dana Hareven, Orna Ben-Naim, Neta Ron, Noam Adir and EliezerArchitecture and Flowering

Tomato SP-Interacting Proteins Define a Conserved Signaling System That Regulates Shoot

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