A New C-Type Cyclin-Dependent Kinase from Tomato Expressed in Dividing Tissues Does Not Interact...

A New C-Type Cyclin-Dependent Kinase from TomatoExpressed in Dividing Tissues Does Not Interact withMitotic and G1 Cyclins1

Jerome Joubes, Martine Lemaire-Chamley, Frederic Delmas, Jocelyne Walter, Michel Hernould,Armand Mouras, Philippe Raymond, and Christian Chevalier*

Unite Mixte de Recherche en Physiologie et Biotechnologie Vegetales (J.J., M.L.-C., F.D., M.H., A.M., P.R.,C.C.) and Unite Mixte de Recherche en Genomique et Developpement des Pathogenes (J.W.), Institut deBiologie Vegetale Moleculaire et Institut National de la Recherche Agronomique, Centre de Recherche deBordeaux, BP 81, 33883 Villenave d’Ornon cedex, France

Cyclin-dependent kinases (CDKs) form a conserved superfamily of eukaryotic serine-threonine protein kinases whoseactivity requires the binding of a cyclin protein. CDKs are involved in many aspects of cell biology and notably in theregulation of the cell cycle. Three cDNAs encoding a C-type CDK, and a member of each B-type CDK subfamily, wereisolated from tomato (Lycopsersicon esculentum Mill.) and designated Lyces;CDKC;1 (accession no. AJ294903), Lyces; CDKB1;1(accession no. AJ297916), and Lyces;CDKB2;1 (accession no. AJ297917). The predicted amino acid sequences displayed thecharacteristic PITAIRE (CDKC), PPTALRE (CDKB1), and PPTTLRE (CDKB2) motives in the cyclin-binding domain, clearlyidentifying the type of CDK. The accumulation of all transcripts was associated preferentially with dividing tissues indeveloping tomato fruit and vegetative organs. In contrast to that of CDKA and CDKBs, the transcription pattern ofLyces;CDKC;1 was shown to be independent of hormone and sugar supply in tomato cell suspension cultures and excisedroots. This observation, together with the absence of a patchy expression profile in in situ hybridization experiments,suggests a non-cell cycle regulation of Lyces;CDKC;1. Using a two-hybrid assay, we showed that Lyces;CDKC;1 did notinteract with mitotic and G1 cyclins. The role of plant CDKCs in the regulation of cell division and differentiation isdiscussed with regard to the known function of their animal counterparts.

The eukaryotic cell cycle is controlled by the or-dered action of a protein complex composed of acatalytic subunit named cyclin-dependent kinase(CDK) and positive regulatory elements named cyc-lins. The association of the CDK with its cyclin part-ner determines the activity of the complex, its stabil-ity, its localization, and substrate specificity (Pines,1994). The complex activity is also regulated by thephosphorylation status of the kinase (Dunphy, 1994),the controlled degradation of the cyclin subunit (Pe-ters, 1998), and the binding of CDK inhibitors orregulatory factors (Lees, 1995).

The complexity of the plant cell cycle is reflected bythe existence of several types of cyclins and CDKs. Sofar, plant cyclins have been classified into five majorgroups: A, B, C, D, and H (Renaudin et al., 1996;Yamaguchi et al., 2000). The A- and B-type cyclinsknown as mitotic cyclins accumulate during the S,G2, and early M phase and during the G2 and earlyM phase, respectively (Mironov et al., 1999). D-typecyclins control the progression through the G1 phasein response to growth factors and nutrients (Riou-

Khamlichi et al., 2000). C- and H-type cyclins havebeen characterized recently in poplar (Populustremula � tremuloides) and rice (Oryza sativa;Yamaguchi et al., 2000). Both of them were foundto interact specifically with the rice CDK-activatingkinase, but only Orysa;CycH;1 could activate thekinase, suggesting that it is the effective regulatorysubunit.

In plants, five distinct classes of CDKs, CDKAthrough CDKE, have been defined according to phylo-genetic, structural, and functional similarities with an-imal and yeast CDKs (Joubes et al., 2000a). This classi-fication is mainly based on the conservation of thePSTAIRE motif in the cyclin-binding domain. TheCDKA class groups functional homologs of the yeastp34cdc2/CDC28 protein displaying the PSTAIRE canoni-cal motif. Their expression and translation patterns areconstitutive during the cell cycle. CDKBs possess adivergent motif: either PPTALRE or PPTTLRE, reflect-ing the existence of two subgroups, CDKB1 andCDKB2, respectively. Because both fail to functionallycomplement temperature-sensitive mutants of yeastCDC2/CDC28, they represent plant-specific CDKs.CDKAs are supposed to regulate both the G1-S andG2-M transitions, whereas CDKBs regulate the G2-Mtransition (Mironov et al., 1999). The three other CDKfamilies (CDKC, D, and E) representing non-PSTAIREkinases are poorly characterized and their function in

1 This work was supported by the Region Aquitaine and theAction Incitative Programmee Agraf-Institut National de la Re-cherche Agronomique (“Elaboration de la Qualite des Fruits”).

* Corresponding author; e-mail [email protected]; fax33–556 – 84 –32– 45.

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the cell cycle regulation remains unclear. However,CDKDs were identified as CDK-activating kinases thatactivate the CDK/cyclin complexes by phosphorylationof the Thr residue within the T-loop region of CDKA(Yamaguchi et al., 1998).

Plant CDKCs display a PITAIRE motif in thecyclin-binding domain. So far, they have been foundin alfalfa (Medicago sativa), pea (Pisum sativum), andArabidopsis (Feiler and Jacobs, 1991; Newman et al.,1994; Magyar et al., 1997); no precise function couldbe assigned. In mammals, the PITAIRE motif identi-fies CDC2-related kinases such as the humancholinesterase-related cell division (CHED) kinase,which is required during hematopoieisis (Lapidot-Lifson et al., 1992). The CDK9 kinase displaying aPITALRE motif also belongs to this group. Its func-tion has been well documented in human because theCDK9 kinase activity is involved in the regulation ofgene transcription elongation (De Falco and Gior-dano, 1998; Bregman et al., 2000).

Early development of tomato (Lycopsersicon esculen-tum Mill.) fruit offers an interesting model for study-ing plant organogenesis, particularly the regulationof cell division and cell expansion phenomena thatappear to account for two distinct developmentalphases (Gillaspy et al., 1993). In an attempt to inves-tigate the molecular mechanisms governing fruit or-ganogenesis, we described the involvement of cellcycle genes encoding CDKA as well as mitotic andG1 cyclins, in the spatial and temporal regulation ofmitotic activity in developing tomato fruits (Joubes etal., 1999, 2000b).

In this work, we present the characterization ofnew tomato CDK genes and their putative implica-tion in fruit development. Three full-length cDNAsencoding a C-type CDK and B1- and B2-type CDKswere isolated from tomato fruit cDNA libraries. Weanalyzed the expression patterns of these genes dur-ing fruit development and in response to differenthormone and nutrient regimes using tomato cell sus-pension and in vitro-cultured excised roots. We dem-onstrated that the CDKC protein does not interactwith any of the mitotic and G1 cyclins isolated intomato so far.

RESULTS

Isolation and Characterization of cDNAs EncodingAdditional Tomato CDKs: CDKC, CDKB1, and CDKB2

To isolate fruit-specific cDNAs preferentially ex-pressed during the cell expansion phase of develop-ment (Gillaspy et al., 1993; Joubes et al., 1999), weperformed a reverse northern procedure based on adifferential screen discriminating expanding fruitscDNAs from full-expanded leaves cDNAs. Amongthe selected clones, we identified a 1,827-bp cDNAharboring a complete ORF encoding a 512-aminoacid peptide. The encoded protein displayed a highpercentage of identical residues with CDKCs from

alfalfa and Arabidopsis: 63% with Medsa;CDKC;1(Magyar et al., 1997) and 59% and 64% with Arath;CDKC;1 and Arath;CDKC;2 respectively (Newman etal., 1994). It shared only 30% of identical residueswith the tomato CDKA;1 (Joubes et al., 1999). Theputative tomato CDKC comprised functional regionscharacteristic of CDKs (Fig. 1A): the cyclin-bindingdomain displaying a PITAIRE motif (residues 63–78),the catalytic domain (residues 163–173), and theT-loop region (residues 184–205) containing a Thrresidue at position 199 (corresponding to T161 ofCDKA), which phosphorylation stabilizes the cyclinbinding. Furthermore, the primary sequence exhibitsthe three Arg residues (at positions 71, 164, and 188)involved in the stabilization of the phosphorylated Tloop, and the two phosphorylation sites: T35 and Y36corresponding to the conserved amino acids T14 andY15 of CDKA. From the analysis of the predictedprimary sequence and protein alignments, this cDNAis likely to code for a new C-type CDK from tomato.Thus, it was named Lyces;CDKC;1 according to theproposed plant CDK gene nomenclature (Joubes etal., 2000a).

Using sequencing data from the The Institute forGenomic Research (Rockville, MD) Tomato Gene In-dex, we could identify two expressed sequence tags(ESTs) corresponding to cDNAs encoding respec-tively a member of the CDKB1 (EST 326331) and theCDKB2 subfamily (EST 336587). The correspondingfull-length cDNAs were isolated after screening the“young tomato fruit” cDNA library (Joubes et al.,1999). The analysis of the predicted amino acidsequences (Fig. 1A) revealed the presence of thePPTALRE and PPTTLRE motives characterizingCDKB1 and CDKB2 proteins, respectively (Joubes etal., 2000a). The two cDNAs thus were named Lyces;CDKB1 and Lyces;CDKB2. The sequence of their re-spective encoded proteins shared 60.7% of identicalresidues, and only 46.2% and 44.4% with that ofCDKA;1. The amino acid sequence of Lyces;CDKC;1isolated herein showed a lower percentage of iden-tity with these two B-type CDKs (25% with CDKB1and 32% with CDKB2).

The identity of the tomato CDKC, CDKB1, andCDKB2 was confirmed by a phylogenetic analysisusing the multiple alignment program CLUSTAL W(Fig. 1B). Lyces;CDKC;1 was classified clearly intothe cluster containing the plant CDKCs (ms;c1,Medsa;CDKC;1; at;c1, Arath;CDKC;1; and at;c2,Arath;CDKC;2) distantly from the other clusters ofplant CDKs. Within the same cluster, two otherbranches of the tree can be defined, displaying re-spectively the animal PITAIRE CDKs (human hs;CHED, accession no. Q14004, hs;CrkRs, accession no.AF227198, and Caenorhabditis elegans ce;9, accessionno. P46551) and PITALRE CDK9 (human hs;9, acces-sion no. P50750, and Drosophila melanogaster dm;9,accession no. AAB84112).

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Expression of CDKC and CDKB Genes during FruitDevelopment and in Plant Organs

The relative transcript levels corresponding to thedifferent tomato CDK genes were analyzed by semi-quantitative reverse transcriptase (RT)-PCR using acombination of primers located in the coding se-quence and in the 5�- or 3�-untranslated region se-quence of the cDNAs, respectively (Table I). Specificprimers for an actin cDNA were used as an internalcontrol of RT-PCR.

In the course of fruit development (Fig. 2A), theLyces;CDKC;1 transcripts were highly expressed atanthesis (A) and up to 5 DPA, i.e. during the celldivision phase. After 5 DPA, their abundance de-creased to reach a basal level during the cell expan-sion phase (8–20 DPA) and until the onset of ripening(MG stage). The expression profiles of Lyces;CDKC;1and Lyces;CDKA;1 were similar, although the rela-tive amount of Lyces;CDKA;1 transcripts was higher.Lyces;CDKB1;1 and Lyces;CDKB2;1 were also highlyexpressed from anthesis to 5 DPA. Their expressiondecreased abruptly at 8 DPA and almost disappearedat the MG stage. During fruit development, the ex-pression of the actin cDNA was found to be consti-tutive. The transcript levels for these four tomatoCDKs as well as the actin cDNA were enhanced in allplant organs displaying meristematic activity such asyoung leaves and roots. In stems, the level of CDKCand the two CDKB transcripts was very low. In full-differentiated leaves, i.e. in nondividing tissues, avery weak expression signal could be observed forLyces;CDKC;1, whereas Lyces;CDKB1;1 and Lyces;CDKB2;1 transcripts were undetectable.

We previously demonstrated that the distributionof mitotic activity is not only temporally but alsospatially regulated in the tomato fruit (Joubes et al.,1999). Cell division occurs in the epidermis until theend of development, whereas the inner pericarp andgel (locular tissues) cells are characterized by an ar-rest of mitosis after 15 DPA and the concomitantendoreduplication of the nDNA content. Therefore,we investigated the expression of tomato CDK genesin the different tissues of the developing fruit (Fig.2B). In the epidermis, the CDKC and actin transcriptsappeared to be constitutively expressed between 10DPA and the RR stage, whereas CDKA transcriptsaccumulated until the MG stage. Lyces;CDKB1;1 andLyces;CDKB2;1 were weakly expressed when com-pared to the other CDK genes. However a longerexposition of the autoradiography showed similarpatterns of expression than that of CDKA (data notshown). In the pericarp, mRNAs corresponding toCDKC and CDKA accumulated up to 20 DPA, thendecreased to almost undetectable levels at the RRstage. Lyces;CDKB1;1 was weakly but constantly ex-pressed between 10 DPA and the MG stage. Lyces;CDKB2;1 was expressed between 10 and 15 DPA, andthen became barely detectable. The actin gene wasalmost constituvely expressed between 10 DPA and

the MG stage, and then its expression decreased atthe maturation stage. In the gel tissue, Lyces;CDKC;1was always expressed during development, withvery high levels between 10 and 15 DPA. The expres-sion of Lyces;CDKA;1 was found to be constitutiveduring the gel development up to the MG stage. Thegenes encoding the tomato CDKB1 and CDKB2 weremainly expressed up to 15 DPA and almost disap-peared thereafter. The transcript level for the actincDNA decreased gradually in the course of the geldevelopment.

Lyces;CDKC;1 Is Expressed in Floral Meristem andYoung Fruits

To deepen the analysis of the temporal and spatialgene expression patterns, the accumulation of Lyces;CDKC;1 transcripts was further investigated bymRNA in situ hybridization (Fig. 3). In flower buds1.5 to 2 mm long, a weak signal was uniformly dis-tributed in all tissues (Fig. 3A). However, a strongersignal was observed in the primordia of petals andstamens, and in the L1 cell layer (Fig. 3A, inset). Infruits harvested at anthesis, a transient accumulationof the transcripts was detected in the pericarp, theplacenta, and the ovules (Fig. 3C). At a higher mag-nification, the hybridization signal was observedclearly in the epidermis and subepidermis cell layersof the pericarp, i.e. the exocarp and the endocarp(Fig. 3E). In ovules, Lyces;CDKC;1 transcripts weredetected in the cell layers of the integument and thenucellus. Then the hybridization signal decreasedgradually in 5-DPA fruits (Fig. 3G).

The Expression of Lyces;CDKC;1 Is Not Controlled bySugar and Phytohormones

To investigate the influence of sugar and phytohor-mones on the transcription level of the different CDKgenes, we used tomato cell suspension cultures (Fig.4A). When cells were incubated without Glc, BAP,and 2,4-D (Control 2), the levels of transcripts forCDKC, CDKBs, and CDKA were greatly reducedcompared with un-starved cells (Control 1). Aftergrowth under various regimes of sugar and hormonecombinations, the level of Lyces;CDKC;1 mRNAs wasenhanced when compared with Control 2, thus sug-gesting that sugar starvation has little effect on theLyces;CDKC;1 transcription. The refeeding of thecells with Glc induced the transcription of the CDKAand CDKB genes, independently of the hormonalstatus. However, after 72 h of sugar starvation, theaddition of one or both hormones to Glc-free mediumdid not induce the transcription of CDKA and CDKBgenes, which remained similar to that in Control 2.Sugar starvation exerted a similar and drastic effecton the accumulation of actin transcripts as previouslyobserved by Sheu et al. (1994).

CDKC and CDKB Gene Expression in Tomato Fruit Development

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Figure 1. (Legend appears on facing page.)

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The ability of sugars to control the expression ofthe CDK genes was further analyzed using excisedtomato roots submitted to sugar starvation (Fig. 4B).The expression of Lyces;CycA2;1 and Lyces;CycD3;1cDNAs encoding a CycA2 mitotic cyclin and aCycD3 G1 cyclin, respectively (Joubes et al., 2000b)was also investigated. In agreement with the resultsobtained with cell suspensions (Fig. 4A), the level ofCDKC transcripts in excised tomato roots waspoorly affected by Suc starvation because it remainsconstant during the whole starvation period (Fig.4B). The transcripts for CDKA and the two CDKBs,as well as actin, were rapidly and strongly affectedby sugar starvation. The accumulation of the CycA2and CycD3 cyclin transcripts were impaired simi-larly because they became almost undetectable af-ter4 d of starvation. The addition of Suc back to themedium after 1 or 3 d of starvation induced thetranscription of all the genes tested. It is interestingthat when Suc was added back to the medium, theCDKC gene transcription was induced comparedwith the level of transcripts in the control corre-

sponding to excised untreated roots, thus suggest-ing a positive effect of sugars on the CDKC genetranscription.

Lyces;CDKC;1 Does Not Interact with TomatoMitotic and G1 Cyclins

To examine whether Lyces;CDKC;1 can interactwith the already known tomato cyclins, a yeast two-hybrid approach was used. The putative protein-protein interactions between the different tomatocDNAs encoding CDKs (CDKA1, CDKA2, CDKB1,CDKB2, and CDKC; Joubes et al., 1999; this work)and cyclins (Lyces;CycA1;1, Lyces;CycA2;1; Lyces;CycA3;1, Lyces;CycB2;1, and Lyces;CycD3;1; Joubeset al., 2000b) were tested (Table II). To increase thestability of the putative complexes formed in theyeast cell, we used constructs in which the destruc-tion box and PEST domain of the mitotic cyclins andthe D-type cyclin, respectively, were deleted. Theexpression of fusion proteins between the GAL4 DBDand the different CDKs on one hand, and the GAL4

Table I. Sets of PCR primers used to amplify gene-specific regions and corresponding size of the amplified product

Gene Primer Sequence (5� 3 3�) Location Expected Size

bp

Lyces;CDKA;1Sense GCT TAT TGT CAT TCT CAT AGA GTT CTT 479–505 559Antisense CTG GAT GAA GGG GCA GAC AAT CAC GG 1,013–1,038

Lyces;CDKB1;1Sense GCT AAA CCA ATC AAT CAA GC 61–80 415Antisense CTA TGG CTG TGA GCA ACC CC 457–476

Lyces;CDKB2;1Sense GGA GGC TGC TGA AAA TGC TG 46–65 521Antisense GTA TAA GCT CTG CCA AGT CC 548–567

Lyces;CDKC;1Sense GCA CCC ACA GCA ACA TTC GCG CCT CCC TCC 1,178–1,207 618Antisense TAT ACC TAA GCC AAG AAA TAT CTA CCG 1,770–1,796

Lyces;CycA2;1Sense TAT GAA GAA ATT TGT GCA CCT CGT G 1,208–1,235 554Antisense GGA TTG GCC ACC GAG ACT TAA AAT CAG C 1,735–1,762

Lyces;CycD3;1Sense TTA TCT TTC ATT GAT CAT ATT ATG AGG 772–798 525Antisense CTA GGT AAT CTA GAG AAC AAG ATA TCG 1,271–1,297

ActinSense TGG CAT CAT ACT TTC TAC AAT G 323–344 615Antisense CTA ATA TCC ACG TCA CAT TTC AT 915–938

Figure 1. Comparison of amino acid sequences of CDKs. A, Multiple alignment of the deduced amino acid sequences oftomato CDKs (Lyces;CDKC;1, AJ294903; Lyces;CDKB1;1, AJ297916; Lyces;CDKB2;1, AJ297917; and Lyces;CDKA;1,Y17225), alfalfa CDKC (Medsa;CDKC;1, T50815), and the two Arabidopsis CDKCs (Arath;CDKC;1, T09572 and Arath;CDKC;2, BAA97308). Deduced amino acid sequences of the different CDKs were compared using the multiple alignmentprogram CLUSTAL W version 1.7. The identical residues between Lyces;CDKC;1 and the different CDKs are represented bydots. Residues essential for activity are indicated by stars. Positions of the cyclin-binding domain (I), the catalytic domain(II), and the T-loop region (III) are boxed in black. Gaps (�) were introduced for maximizing the alignments. B, Phylogeneticanalysis of CDKs. The most conserved part of the CDK core (from residue 11–276 of CDKAs) was compared using themultiple alignment program CLUSTAL W version 1.7. The Phylip package was used for the construction of the phylogenetictree and comparison of parsimony and distance (Neighbor Joining algorithm) methods. The robustness of the tree wasassessed by the bootstrap method on 1,000 replicates. The length of lines is proportional to the genetic distance betweeneach node. Within the tree, the species are indicated by the first letters of the generic and specific names as described byJoubes et al. (2000a). Tomato CDKB1, CDKB2, and CDKC are highlighted in gray.





AD and the different cyclins on the other hand, wasconfirmed by western blot using anti-LexA and anti-Gal4 antibodies, respectively (data not shown). Theinteraction between the two GAL4 domains was re-vealed by the growth on a medium lacking His(His�) and by measuring the �-galactosidase activ-ity resulting from the activated transcription of thelacZ gene.

As shown in Table II, the two CDKAs interactedvery efficiently with CycA2, and to a lower extentto CycD3. They were unable to interact with CycA1,CycA3, and CycB2. CDKB2 interacted also withCycA2 but not with other kinds of cyclins. No inter-action could be observed between CDKC or CDKB1and any of the tested cyclins, suggesting the existenceof other types of cyclin protein able to interact withthese CDKs.

DISCUSSION

Three cDNAs from tomato, named Lyces;CDKC;1,Lyces;CDKB1;1, and Lyces;CDKB2;1, representingnew CDKs were isolated and their expression pat-terns in the course of early fruit development andunder different nutritional regimes investigated.

Based on the characterization of their amino acidsequence and according to a phylogenetic analysis(Fig. 1), we identified Lyces;CDKC;1 as a member ofthe C-type CDK family characterized by the presenceof a PITAIRE motif in the cyclin-binding domain, andLyces;CDKB1;1 and Lyces;CDKB2;1 as members ofdifferent subfamilies of mitotic CDKBs (Joubes et al.,2000a). Analysis of the The Institute for GenomicResearch Tomato Gene Index allowed the identifica-tion of two ESTs encoding CDKC: The first one (EST401472) corresponds to Lyces;CDKC;1 isolated in

Figure 2. Expression analysis of CDK genes in tomato. A, Semiquantitative RT-PCR analysis of CDK gene expression duringfruit development and in vegetative organs. Total RNA to be used for RT was isolated from fruits harvested at the followingdevelopmental stages: anthesis (A), 3, 5, 8, 10, 15, and 20 DPA, and mature green (MG) stage, and from young leaves (YL),differentiated leaves (DL), roots (Ro), and stems (St). The specific amplification of cDNA fragments was detected after gelelectrophoresis, Southern blotting, and hybridization to the corresponding 32P-labeled probes. B, Semiquantitative RT-PCRanalysis of CDK gene expression in the different fruit tissues during development. Total RNA was isolated from dissected fruittissues (epidermis, pericarp, and gel) at the following developmental stages: 10, 15, and 20 DPA; MG and red ripe (RR)stages.

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this work and the second one (EST 307422) to anew CDKC. A genomic Southern-blot analysis usingLyces;CDKC;1 confirmed the existence of at least twogenes in tomato (data not shown). The predictedtranslation products of these two tomato CDKCgenes, as well as those deduced from the only twogenes identified in the Arabidopsis genome, are char-acterized by the presence of solely a PITAIRE motif.This suggests that, unlike animal CDKCs such asCDK9, the PITALRE motif may not be found in theplant CDKC family. It is interesting that the twobranches of the phylogenetic tree clustering animalPITAIRE and PITALRE CDKs seem to diverge afterthe formation of the plant CDKC cluster (Fig. 1B).Hence, this could imply that the plant CDKC group

may contain indifferently functional homologs ofboth the animal PITAIRE and PITALRE CDKs.

The isolation of these new CDKC, CDKB1, andCDKB2 from tomato allowed us to investigate howtheir gene expression may be developmentally regu-lated during fruit organogenesis, and especially todetermine their respective involvement in the celldivision process. Cell division plays indeed a crucialrole during fruit organogenesis because the numberof cells resulting from mitotic activity is an essentialdeterminant of the final size and sink strength of thefruit (Gillaspy et al., 1993; Frary et al., 2000). Earlydevelopment of tomato fruit can be divided into twodistinct phases (Gillaspy et al., 1993). The first onelasting for about 7 to 10 d after fertilization and fruit

Figure 3. Analysis of Lyces;CDKC;1 transcriptaccumulation by in situ hybridization. Longitu-dinal sections of early stage flower buds (1.5–2mm in size) and fruits harvested at anthesis and5 DPA were hybridized with digoxigenin-labeled RNA probes. A and B, Section of floralbud; C and D, section of a fruit at anthesis; E andF, higher magnification of the section of a fruit atanthesis; G and H, section of a fruit at 5 DPA. A,C, E, and G were hybridized with the antisenseprobe; B, D, F, and H were hybridized with thesense probe (negative controls). en, Endosperm;ep, epidermis; in, integument; nu, nucellus; ov,ovule; pe, pericarp; pl, placenta; pp, petal pri-mordium; se, seed; sp, stamen primordium. Barscale � 100 �m.





set is characterized by a very active period of celldivisions inside the ovary. During the second phase,fruit growth is mostly due to cell expansion, thusleading to a fruit that exhibits its almost final size andis able to ripen. Inside the developing tomato fruit, thedistribution of mitotic activity is spatially and tempo-rally regulated in relationship with the differentialexpression of CDKA and cyclins (Joubes et al., 1999,2000b). At the transcriptional level, we alreadyshowed that the CDKA expression was associatedwith dividing tissues but also with expanding tissues(Joubes et al., 2000b). In the course of fruit develop-ment and in vegetative organs, the overall expression

pattern of Lyces;CDKC;1, Lyces;CDKB1;1, and Lyces;CDKB2;1 were similar to that observed for Lyces;C-DKA;1, i.e. a higher level of transcripts in dividingorgans displaying meristematic activity (Fig. 2A). Atthe spatial level, the in situ hybridization experimentconfirmed the preferential expression of Lyces;CDKC;1 in dividing tissues (e.g. floral organ primor-dia and ovules; Fig. 3). A uniformly distributed signalwas observed in these dividing tissues, but clearlyLyces;CDKC;1 does not display a patchy pattern ofexpression as expected for a cell cycle-regulated gene.

It is interesting that a marked difference in theexpression pattern of Lyces;CDKB1;1 and Lyces;

Figure 4. Effect of hormones and sugars on CDKgene expression. A, Semiquantitative RT-PCRanalysis of CDK gene expression in cell suspen-sions under the control of nutrient and hormonalfactors. Freshly diluted tomato cell suspensioncultures were grown for 72 h in a completemedium and subsequently depleted of auxin,cytokinin, and Glc. After depletion for 72 h, thecells were subdivided and grown in the pres-ence (�) or absence (�) of Glc (G),6-benzylaminopurine (BAP; B), and 2,4-dichlorophenoxyacetic acid (2,4-D; D). Twenty-four hours later, the cells were harvested. Con-trols 1 and 2 represent cells harvested beforedepletion and 72 h after depletion respectively.B, Semiquantitative RT-PCR analysis of CDKgene expression in excised tomato roots submit-ted to Suc starvation. Tomato roots were excised(control C) and cultivated in liquid medium inthe presence (�) or absence (�) of Suc during4 d. Starved roots were refed with Suc during24 h after 1 or 3 d of starvation.

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CDKB2;1 was observed in differentiated leaves as thetranscripts completely disappeared (Fig. 2A). Thisresult is in accordance with the typical function ofCDKBs in dividing cells (Mironov et al., 1999; Joubeset al., 2000a). This was further confirmed by analyz-ing the CDK gene expression in the different tissuesof developing fruits, and especially in the gel tissue(Fig. 2B). After 15 DPA, the cells composing the gelstop dividing and expand concomitantly with nDNAendoreduplication (Joubes et al., 1999). Because theendoreduplication cycle is made of the succession of

S and G phases without mitosis (for review, seeJoubes and Chevalier, 2000), the expression of Lyces;CDKB1;1 and Lyces;CDKB2;1 was strongly reduced.A very faint signal could still be observed up to theMG stage for Lyces;CDKB1;1, whereas Lyces;CDKB2;1 transcripts became undetectable after 15DPA. This slight difference in the gene expressionmay originate from the S/G2 and G2/M phase de-pendence at the transcriptional level of CDKB1 andCDKB2, respectively (Mironov et al., 1999; Meszaroset al., 2000). In Arabidopsis, the only CDKB gene so

Table II. Interactions between tomato CDKs and the deleted forms of tomato cyclins in yeast

Protein Fused to

His� Growtha His� Growthb �-GalactosidasecDNA-binding domain

(DBD)Activating domain

(AD)

CDKA1 CycA1;1 � � –CDKA1 CycA2;1 � � 63.3 � 1.9CDKA1 CycA3;1 � � –CDKA1 CycB2;1 � � –CDKA1 CycD3;1 � � 9.7 � 3.6CDKA1 – � � –

CDKA2 CycA1;1 � � –CDKA2 CycA2;1 � � 45 � 2.1CDKA2 CycA3;1 � � –CDKA2 CycB2;1 � � –CDKA2 CycD3;1 � � 23.3 � 6.9CDKA2 – � � –

CDKB1 CycA1;1 � � –CDKB1 CycA2;1 � � –CDKB1 CycA3;1 � � –CDKB1 CycB2;1 � � –CDKB1 CycD3;1 � � –CDKB1 – � � –

CDKB2 CycA1;1 � � –CDKB2 CycA2;1 � � 5.5 � 2.3CDKB2 CycA3;1 � � –CDKB2 CycB2;1 � � –CDKB2 CycD3;1 � � –CDKB2 – � � –

CDKC CycA1;1 � � –CDKC CycA2;1 � � –CDKC CycA3;1 � � –CDKC CycB2;1 � � –CDKC CycD3;1 � � –CDKC – � � –

laminin �1 CycA1;1 � � –laminin �1 CycA2;1 � � –laminin �1 CycA3;1 � � –laminin �1 CycB2;1 � � –laminin �1 CycD3;1 � � –laminin �1 – � � –

RAS RAF � � 78.1 � 2.5a Ability of transformed yeast cells to grow on selection plates containing His (His�). b Ability of

transformed yeast cells to grow on selection plates lacking His (His�). c �-Galactosidase activitywas measured only for positive interactions (indicated as Miller units).





far studied, CDC2b, corresponds to a member of theCDKB1 family (Arath;CDKB1;1, Joubes et al., 2000a).This gene was found recently to play a role in regu-lating seedling growth in darkness independent ofcell division or endoreduplication (Yoshizumi et al.,1999), thus suggesting that Arath;CDKB1;1 may beinvolved in regulating directly hypocotyl cell elon-gation or a specific phase of the cell cycle and/oroverall chromosome spatial organization, critical forhypocotyl cell elongation and cotyledon develop-ment. These findings agree with the lack of partici-pation in the promotion of endoreduplication wecould also observe for Lyces;CDKB1;1 in the gel (Fig.2B). Because Lyces;CDKB1;1 is still expressed at a lowlevel in expanding cells of the gel, it may play a rolein the regulation of cell expansion as suggested byYoshizumi et al. (1999), implying a striking differencein the respective function of Lyces;CDKB1;1 andLyces;CDKB2;1.

In the gel tissue, the Lyces;CDKC;1 transcripts ac-cumulated at high levels in dividing cells (until 15DPA). When only endoreduplication occurs (after 15DPA), the accumulation of transcripts dropped to abasal level. Lyces;CDKA;1 behaved quite differentlybecause the transcripts were highly expressed duringthe development of the gel tissue at very high levelsto the onset of ripening. Hence, it is tempting tosuggest a role for CDKA in the regulation of theendocycle (Joubes and Chevalier, 2000).

Very little is known about the control of plantCDKC gene expression. Because sugar and hormoneshave a role of prime importance during the process offruit development (Gillaspy et al., 1993), we investi-gated the influence of these parameters on the regu-lation of Lyces;CDKC;1 transcription. The gene ex-pression of Lyces;CDKC;1 was only slightlymodulated by sugars or hormones (Fig. 4A). Thetranscription of Lyces;CDKA;1 was induced by thepresence of Glc, whereas that of Lyces;CDKB1;1 andLyces;CDKB2;1 were strictly dependent upon sugaravailability. Such results dealing with the nutritionalcontrol of CDKA and CDKB gene transcription are inagreement with those described for Arabidopsis cellsuspension by De Veylder et al. (1999). In excisedtomato roots (Fig. 4B), even after 4 d in the absence ofSuc, the Lyces;CDKC;1 gene expression was not af-fected by Suc starvation, whereas the CDKA andCDKB gene expressions were deeply affected, as wellas those for mitotic cyclin CycA2;1 and G1 cyclinCycD3;1 used as controls of cell cycle- and sugar-regulated genes (Riou-Khamlichi et al., 1999, 2000;Burssens et al., 2000). The observation that Lyces;CDKC;1 expression is not modulated by nutritionalor hormonal parameters and the constitutive pres-ence of transcripts in cells that have stopped to di-vide suggests that the CDKC gene expression is notcell cycle-regulated. This is supported by the absenceof a patchy pattern in the in situ hybridization exper-iments (Fig. 3), in tissues displaying an intense cell

division activity, such as floral primordia, develop-ing ovules, and very young fruits. Moreover, it wasdemonstrated that the expression of CDKC tran-scripts was constitutive throughout the cell cycle inalfalfa synchronized cells (Magyar et al., 1997), andthat the gene expression of the human CDK9 kinasewas not cell cycle-regulated and its kinase activitydid not vary appreciably during the cell cycle (Breg-man et al., 2000).

The next question we assessed was to determinewhether Lyces;CDKC;1 could interact with the dif-ferent cyclin partners available in tomato (Joubes etal., 2000b). Using the yeast two-hybrid system, dif-ferent authors demonstrated the protein-protein in-teractions between CDKA or CDKB and variousD-type cyclins (De Veylder et al., 1997, 1999; Nak-agami et al., 1999; Meijer and Murray, 2000; Meszaroset al., 2000), CDKA and CycA2 (Roudier et al., 2000),and CDKD and its specific partners CycH and CycC(Yamaguchi et al., 2000). However, no exhaustiveanalysis of plant CDK/cyclin binding has been per-formed so far. Here, we were able to test a combina-tion of 25 different CDK/cyclin interactions (TableII). Like in other plant species, we demonstrated thattomato CDKA interact with CycA2 and CycD3;1, butnot with other mitotic cyclins (CycA1, CycA3, andCycB2). Moreover, the tomato CDKB2 could bindCycA2. In our hands, CDKB1 failed to interact withany of the tested cyclins, unlike the alfalfa CDKB1,which binds a cyclin D3 (Meszaros et al., 2000). Wewere unable to reveal any interaction between Lyces;CDKC;1 and mitotic and G1 cyclins. Therefore, thecyclin partner of plant CDKC remains to be identifiedamong the numerous different cyclin genes presentin a plant genome (e.g. more than 30 in Arabidopsis).

The function of CDKC in plant cells remains to beelucidated. In mammals, the function of PITAIREkinases (such as the CHED kinase) is unknown.However the second type of CDKC-related kinases(CDK9) received much more attention as the kinaseactivity is associated with protein complexes impli-cated in HIV gene transactivation (Romano et al.,1999). Human CDK9 binds cyclins T and K, leadingto the formation of different complexes with specificactivity (Bregman et al., 2000). The CDK9/CycT com-plex, called the positive-transcription elongation fac-tor b, possesses a carboxyl-terminal domain (CTD)kinase activity which was shown to activate the genetranscription in vivo (Napolitano et al., 2000). TheCTD phosphorylation of the RNA polymerase II larg-est subunit by positive-transcription elongation fac-tor b enables the polymerase to promote the tran-scription elongation by counteracting the effect ofnegative factors. Hence, CDK9 is a multifunctionalkinase involved in the control of cell growth and/orcellular viability, even if its activity is not cell cycle-regulated (De Falco and Giordano, 1998). Assumingthat Lyces;CDKC;1 may represent a plant orthologueof CDK9, it is tempting to hypothesize that its pref-

Joubes et al.




erential gene expression in actively dividing cellsrather than in expanding/endoreduplicating cells,could be associated with a putative CTD kinase ac-tivity responsible for the control of gene transcrip-tion. This putative function of Lyces;CDKC;1 mayexplain the intense signal of hybridization observedin floral primordia and developing ovules (Fig. 3). Itmay reflect the high transcription activity needed forthe development of primordia and ovules becausethese tissues undergo an intense cell division activityand specific cell differentiation processes.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Cherry tomato (Lycopersicon esculentum Mill. cv WestVirginia 106) plants and tomato cells derived from thepericarp of tomato (cv Sweet 100) fruit (Jean-Luc Montillet,Commissariat a l’Energie Atomique, Cadarache, France)were cultured as described by Joubes et al. (2000b). Cellswere grown in modified Murashige and Skoog medium,containing 20% (w/v) Glc, 100 mg mL�1 2,4-D, and 10 mgmL�1 BAP; pH was adjusted to 5.8. Cells were grown in thedark, at 25°C, and shaken at 150 rpm. They were main-tained in exponential growth phase by 5-fold dilution ev-ery 6 d in new medium. Cells were harvested by filtrationon glass wool under vacuum, frozen quickly in liquidnitrogen, and stored at �80°C.

Tomato roots were prepared as follows. Seeds weresoaked in 90% (v/v) ethanol, washed twice in sterile water,and immersed in 2% (v/v) sodium hypochlorite solutioncontaining 0.2% (w/v) SDS for 15 min. Seeds were thenwashed four times in sterile water. The sterilized seedswere let to germinate between two layers of paper (What-man, Maidstone, UK) soaked in the mineral solution A(Saglio and Pradet, 1980) in sterile culture boxes (10 � 10 �10 cm) for 3 d at 25°C and in darkness. After 3 d, the upperlayer of Whatman paper was removed and the culturemedium was replaced by the same solution supplementedwith 0.1 m Suc. The boxes were then transferred to agrowth chamber under the culture conditions as describedby Joubes et al. (2000b). After 4 d, plantlets were harvestedand roots excised under sterile conditions. The roots weresubsequently incubated at 25°C in medium A (Saglio andPradet, 1980) supplemented with an antibiotic-antimycoticmixture (ref. A-7292, 10 �L mL�1; Sigma, Saint QuentinFallavier, France), under a continuous bubbling of a gasmixture containing 50% O2 � 50% N2 gas mixture, and inthe presence or absence of 0.1 m Suc. The incubation me-dium was renewed daily. Excised roots were removed atdifferent times, washed with sterile water, dried on filterpaper, and promptly frozen in liquid nitrogen prior to totalRNA isolation.

Extraction of Total RNA

Total RNA from whole fruits, fruit tissues, or variousorgans of tomato plants was extracted as previously de-scribed (Joubes et al., 1999). Total RNA from tomato cells

was extracted using the RNeasy Plant Mini kit (Qiagen,Courtaboeuf, France). After extraction total RNA fromtomato tissues and cells was dissolved in diethyl pyrocar-bonate-treated water.

cDNA Library Screening

A cDNA library was constructed with poly(A�) mRNAprepared from total RNA extracted from tomato fruitsharvested during the cell expansion phase (between 10–15DPA), as previously described by Joubes et al. (1999). ThecDNA library comprised 6.106 recombinant plaques. Afterplating of the amplified cDNA library, 300 individualizedbacteriophage plaques were picked randomly. The cDNAinserts were PCR amplified from the phage suspensionsusing the pBluescript universal M13-20 and reverse prim-ers, separated after agarose gel electrophoresis, and blottedonto a Hybond N� membrane (Amersham Pharmacia Bio-tech, Les Ulis, France). To screen for fruit-specific cDNAs,a reverse northern procedure was performed using com-plexed probes to hybridize duplicate membranes. As apositive probe, 32P-labeled cDNAs were synthesized asdescribed by Zegzouti et al. (1997) using total RNA fromexpanding fruits (15 DPA), and as a negative probe 32P-labeled cDNAs using total RNA from full-expanded leaves.Clones showing a signal only or a stronger signal with thefruit probe were selected and rescued from the Uni-ZAPXR vector using the R408 helper phage following the man-ufacturer’s instructions (Stratagene, La Jolla, CA). The com-plete nucleotide sequence of the inserts was determinedallowing the isolation and identification of the cloneLyces;CDKC;1.

To isolate cDNAs encoding B-type CDKs, specific probeswere generated by RT-PCR using total RNA from fruits at3 DPA. The specific oligonucleotides used to amplify thecDNAs for B1-type and B2-type CDKs were as indicated inTable I. After cloning into pGEM-T vector (Promega, Lyon,France), the identity of the amplified cDNA fragments wasconfirmed by nucleotide sequencing. The amplified frag-ments were then used as 32P-labeled probes to screen300,000 plaques from the “young fruit” cDNA library(Joubes et al., 1999). After three rounds of screening, thepositive clones were isolated, rescued from the Uni-ZAPXR vector and the complete nucleotide sequence of theinserts was determined.

Estimation of Relative Transcript Levels with RT-PCR

To determine specifically the relative transcript levels ofeach cDNA, RT-PCR assays were performed as previouslydescribed (Joubes et al., 1999, 2000b). For excised tomatoroots, 5 �g of total RNA was used in the RT reaction, anda 10-fold dilution of the generated cDNAs was used sub-sequently in the PCR reaction. The specific sets of primersused for the amplification of each cDNA are summarizedin Table I.

In Situ Hybridization

Flower buds and tomato fruits of different developmen-tal stages were fixed in 2% (v/v) formalin, 5% (v/v) acetic





acid, and 50% (v/v) alcohol for 4 h at room temperature.After fixation the tissues were dehydrated in ethanol seriesand ethanol was subsequently replaced by Histosol Plus(Life Sciences International, Cergy-Pontoise, France). Thetissues were embedded in paraffin according to standardprocedures. Sections (8 �m) were fixed in 3-aminopropyl-triathoxysilan-coated slides, deparaffinised in HistosolPlus. The slides were dehydrated in ethanol series for 5 mineach time and air dried.

The Lyces;CDKC;1 DNA used for riboprobe synthesiswas amplified by PCR using a specific set of primers (TableI) and cloned into the pGEM-T vector (Promega). The senseand antisense digoxygenin-labeled riboprobes were gener-ated by runoff transcription using T7 and SP6 RNA poly-merases according to the manufacturer’s protocol (RocheDiagnostics, Meylan, France). For hybridization the sec-tions on the pre-treated slides were incubated with probeslabeled with digoxigenin-11-rUTP using the digoxigeninnucleic acid labeling kit (Roche Diagnostics) in 100 �L ofthe following mix: 50% (v/v) deionized formamide, 300mm NaCl, 1 mm EDTA, 1� Denhardt’s, 10% (w/v) dextransulfate, and 10 mm Tris-HCl, pH 7.5. The slides werecovered with coverslips, placed in a humid chamber, andincubated for 16 h at 45°C. After hybridization coverslipswere removed and slides washed twice for 5 min in 2�sodium chloride/sodium phosphate/EDTA at room tem-perature. The slides were then washed twice for 5 min in0.1� sodium chloride/sodium phosphate/EDTA at 55°C.Immunological detection of the hybridized probes wascarried out using digoxigenin nucleic acid detection kit(Roche Diagnostics). The color reaction was stopped bywashing the slides in water. Slides were air dried andsections were mounted in Neo-Entellan (Merck, Darm-stadt, Germany).

Two-Hybrid Detection of Protein-Protein Interactions

The coding sequences of the different cDNAs were spe-cifically amplified using Pfu DNA polymerase (Promega)and the resulting cDNAs were cloned into pCR4Blunt-TOPO (Invitrogen, Groningen, The Netherlands). Theywere subcloned into the yeast shuttle plasmids pLexAcontaining the DBD (Vojtek et al., 1993) and pGAD3S2Xcontaining the AD, a modified version of pGAD1318 (Be-nichou et al., 1994). The recombinant plasmids were am-plified in Escherichia coli, purified, and then introduced inthe Saccharomyces cerevisiae strain L40 (Le Douarin et al.,1995) by sequential transformation using lithium acetate(Gietz and Woods, 1994). L40 recombinant cells were se-lected on plates containing 5 mm 3-amino-1,2,4-triazol lack-ing Leu and Trp for double transformants or lacking Leu,Trp, and His to reveal the protein-protein interactions. Thehuman RAS and RAF coding sequences (Vojtek et al., 1993)were cloned into pLexA and pGAD3S2X, respectively, andused as a positive control of interaction. As a negativecontrol of interaction, we used the murine laminin �1 se-quence (Chang et al., 1996) cloned into pLexA in combina-tion with the different recombinant pGAD3S2X. The quan-titative �-galactosidase assays were performed accordingto Urcuqui-Inchima et al. (1999).

ACKNOWLEDGMENT

We would like to thank Carine Devaux for preparingand providing the tomato roots samples.

Received February 12, 2001; returned for revision April 4,2001; accepted May 1, 2001.

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A New C-Type Cyclin-Dependent Kinase from Tomato Expressed in Dividing Tissues Does Not Interact...

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