The Tobacco A-Type Cyclin, Nicta;CYCA3;2, at the Nexus of Cell Division and Differentiation

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The Plant Cell, Vol. 15, 2763–2777, December 2003, www.plantcell.org © 2003 American Society of Plant Biologists

RESEARCH ARTICLES

The Tobacco A-Type Cyclin,

Nicta;CYCA3;2

, at the Nexus of Cell Division and Differentiation

Yu Yu,

a

Andre Steinmetz,

a

Denise Meyer,

a

Spencer Brown,

b

and Wen-Hui Shen

a,1

a

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Université Louis Pasteur de Strasbourg, 67084 Strasbourg, France

b

Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France

Although most of the components of the cell cycle machinery are conserved in all eukaryotes, plants differ strikingly fromanimals by the absence of a homolog of E-type cyclin, an important regulator involved in G1/S-checkpoint control in ani-mals. By contrast, plants contain a complex range of A-type cyclins, with no fewer than 10 members in Arabidopsis. Wepreviously identified the tobacco A-type cyclin

Nicta;CYCA3;2

as an early G1/S-activated gene. Here, we show that anti-sense expression of

Nicta;CYCA3;2

in tobacco plants induces defects in embryo formation and impairs callus formationfrom leaf explants. The green fluorescent protein (GFP)–Nicta;CYCA3;2 fusion protein was localized in the nucleoplasm.Transgenic tobacco plants overproducing GFP-Nicta;CYCA3;2 could not be regenerated from leaf disc transformation,whereas some transgenic Arabidopsis plants were obtained by the floral-dip transformation method. Arabidopsis plantsthat overproduce GFP-Nicta;CYCA3;2 showed reduced cell differentiation and endoreplication and a dramatically modifiedmorphology. Calli regenerated from leaf explants of these transgenic Arabidopsis plants were defective in shoot and rootregeneration. We propose that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E in animals, in thecontrol of plant cell division and differentiation.

INTRODUCTION

Most development of the mature plant takes place postembry-onically and originates in the activities of small groups of cellscalled meristems. Plant cells are surrounded by a rigid cell walland divide in place, providing an interesting model to addressthe role of cell division and cell differentiation in development. Itis generally accepted that control of the numbers, places, andplanes of cell division, coupled with regulated and coordinatedcellular expansion and differentiation, are critical in organogen-esis during plant development (Meyerowitz, 1997). However,the role of cell division as a causal element in plant morphogen-esis has long been debated. Molecular characterizations of mu-tants defective in morphogenesis have not identified cell cycleregulators as responsible elements (Meyerowitz, 1997; Nakajimaand Benfey, 2002). Nor did modulation of the expression of cellcycle genes, including those that encode cyclin-dependent ki-nases (CDKs) (Hemerly et al., 1995; Porceddu et al., 2001) andsome cyclins (Doerner et al., 1996; Cockcroft et al., 2000), re-sult in abnormal morphology, although in most cases plantgrowth rate was altered. Together, these observations suggestthat intrinsic mechanisms exist that operate throughout the or-gan or organism as a unit to dictate size and shape. The molec-ular basis underlying such intrinsic mechanisms, however, isunclear. In addition, conflicting data exist reporting that ectopic

expression of the Arabidopsis D-type cyclin

Arath;CYCD3;1

(Riou-Khamlichi et al., 1999; Dewitte et al., 2003), the Arabidop-sis and tobacco CDK inhibitor

ICK1/KRP1/KIS1

(Wang et al.,2000; De Veylder et al., 2001; Jasinski et al., 2002), and the cellcycle–specific transcription factor

E2Fa-DPa

of Arabidopsis (DeVeylder et al., 2002) affect regeneration, organ shape, and/orthe entire morphology of Arabidopsis plants.

D-type cyclins are conserved in animals and plants and areproposed to be sensors of growth conditions and to trigger theG1/S transition by activating the RBR/E2F-DP pathway (Gutierrezet al., 2002; Shen, 2002; Trimarchi and Lees, 2002). The RBR/E2F-DP pathway not only regulates the expression of genes re-quired for the G1/S transition and S-phase progression but alsois involved in other developmental processes (Muller et al.,2001; Ramirez-Parra et al., 2003). Observations that the ec-topic expression of

Arath;CYCD3;1

or

E2Fa-DPa

in Arabidopsisresults in hyperplasia in leaves and dramatically affects plantmorphogenesis (De Veylder et al., 2002; Dewitte et al., 2003)strengthen the important role of the CYCD/RBR/E2F-DP path-way in the control of cell proliferation and development. Thefact that the phenotype induced by the ectopic expression of

ICK1/KRP1/KIS1

can be attenuated by the simultaneous ec-topic expression of

Arath;CYCD3;1

(Jasinski et al., 2002;Schnittger et al., 2003; Zhou et al., 2003) indicates that

ICK1/KRP1/KIS1

regulates cell proliferation and plant morphogenesisprimarily through the inhibition of function of the Arath;CYCD3;1-CDK kinase complex.

In addition to D-type cyclins, plants contain a large num-ber of mitotic cyclins, no fewer than 19 in Arabidopsis, belong-

1

To whom correspondence should be addressed. E-mail [email protected]; fax 33-3-88-61-44-42.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.015990.

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2764 The Plant Cell

ing to three A-type (CYCA1, CYCA2, and CYCA3) and twoB-type (CYCB1 and CYCB2) subclasses (Renaudin et al., 1996;Vandepoele et al., 2002). B-type cyclins are expressed within anarrow time window from late G2- to mid M-phase, and the ec-topic expression of both

Arath;CYCB1;1

and

Oryza;CYCB2;2

accelerates root growth (Doerner et al., 1996; Lee et al., 2003).However, this substantial alteration of cell division rate doesnot alter root morphology, suggesting that these B-type cyclinsare not critical in cell fate determination. In contrast to animals,in which only a single A-type cyclin gene is present in inverte-brates and two are present in vertebrates (Nieduszynski et al.,2002), plants hold a higher complexity of A-type cyclins, com-prising in Arabidopsis two A1-type (CYCA1;1 and CYCA1;2),four A2-type (CYCA2;1, CYCA2;2, CYCA2;3, and CYCA2;4),and four A3-type (CYCA3;1, CYCA3;2, CYCA3;3,and CYCA3;4)members (Chaubet-Gigot, 2000; Vandepoele et al., 2002). Insynchronized tobacco BY2 cells, different A-type cyclins areexpressed sequentially at different time points from late G1/early S-phase until mid M-phase (Reichheld et al., 1996). Thealfalfa A2-type cyclin

Medsa;CYCA2;2

is expressed in allphases of the cell cycle, but its associated kinase activity peaksboth in S-phase and during the G2/M transition (Roudier et al.,2000). These molecular data suggest that A-type cyclins mayhave different functions in plants.

We were particularly interested in functional characterizationof the tobacco A3-type cyclin

Nicta;CYCA3;2

because of itsearly activated expression at the G1/S transition (Reichheld etal., 1996). We first regenerated transgenic tobacco plants ex-pressing

Nicta;CYCA3;2

under the control of the tetracycline-inducible promoter. Local and transient induction of

Nicta;CYCA3;2

expression induces cell division in both the shoot apical meri-stem and the leaf primordia (Wyrzykowska et al., 2002). How-ever, significant effects could not be established when the in-duction of expression was performed at the whole-plant level.Recently, it was reported that transgenic alfalfa plants express-ing

Medsa;CYCA2;2

under the control of the constitutive 35Spromoter exhibit high transcript levels but unchanged proteinlevels and a normal phenotype (Roudier et al., 2003).

Here, we investigated the expression of

Nicta;CYCA3;2

in to-bacco plants and found it to be positively associated with pro-liferating tissues. By fusion with the green fluorescent protein(GFP), we demonstrate that the Nicta;CYCA3;2 protein local-ized exclusively in the nucleoplasm with speckle structures. Weaddressed the function of

Nicta;CYCA3;2

in tobacco by anti-sense expression and found that its loss of function induceddefects in embryo formation and impaired callus formation invitro from leaf explants. We failed to obtain transgenic tobaccoplants that overproduce the GFP-Nicta;CYCA3;2 fusion pro-tein, possibly because its high levels are incompatible withplant regeneration from transformed leaf explants. Neverthe-less, using the floral-dip method, we obtained some transgenicArabidopsis plants that showed variable (from undetectable tohigh) levels of fluorescence of the fusion protein. Only trans-genic Arabidopsis plants containing high levels of the fusionprotein showed a dramatically modified phenotype. They had adecreased level of endoreplication (also named endoreduplica-tion) and an increased level of histone transcripts, indicatingthat GFP-Nicta;CYCA3;2 inhibits cell differentiation. Further-

more, calli regenerated from leaf explants of these transgenicArabidopsis plants were defective in shoot and root regenera-tion. By immunoprecipitation and affinity binding assays, weconfirmed that GFP-Nicta;CYCA3;2 can form active CDK com-plexes in Arabidopsis.

RESULTS

Expression of the

Nicta;CYCA3;2

Gene

Four A-type cyclins of tobacco had been characterized previouslyfor their expression pattern during the cell cycle in BY2 suspensioncells (Setiady et al., 1995; Reichheld et al., 1996).

Nicta;CYCA1;1

and

Nicta;CYCA2;1

are expressed from mid S-phase to midM-phase, whereas

Nicta;CYCA3;1

and

Nicta;CYCA3;2

are ex-pressed earlier, from the G1/S transition to early M-phase. Dur-ing the G1/S transition,

Nicta;CYCA3;2

is expressed

�

1 h ear-lier than

Nicta;CYCA3;1

, which precedes the aphidicolin blockagepoint (Reichheld et al., 1996). To gain insight into the function of

Nicta;CYCA3;2

in plant development, we analyzed its expres-sion in different organs of tobacco plants using RNA gel blothybridization (Figure 1).

Nicta;CYCA3;2

mRNA was detected ata high level in flower buds but was barely detectable in leaves,stems, and roots. The absence of a hybridization signal in theselatter organs may be attributable to the limited sensitivity ofRNA gel blot analysis. We believe that the high level of

Nicta;CYCA3;2

mRNA in flower buds is associated primarilywith the presence of proliferating tissues in this organ and toonly a limited degree with flower specificity. This is because,first, a pattern of expression similar to that of

Nicta;CYCA3;2

Figure 1. RNA Gel Blot Analysis of Nicta;CYCA3;2 Transcripts in Dif-ferent Organs of Tobacco Plants.

RNA samples prepared from leaves (L), stems (S), flower buds (FB), androots (R) were hybridized successively with the Nicta;CYCA3;2, the his-tone H4, the ribonucleotide reductase RNR2, and the ribosomal 18SrRNA probes. The ribosomal 18S rRNA probe served as a loading control.

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Nicta;CYCA3;2

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was observed for the histone

H4

and ribonucleotide reductase

RNR2

(Figure 1), the two genes known to be expressed in dif-ferent types of proliferating tissues (Chaubet et al., 1996; Chaboutéet al., 1998), and second, a high level of

Nicta;CYCA3;2

mRNAwas detected in young seedlings (see Figure 4A). In addition,

Nicta;CYCA3;2

transcripts were detected previously by in situhybridization in leaf primordia (Wyrzykowska et al., 2002).

Subcellular Localization of the Nicta;CYCA3;2 Protein

To study the localization of the Nicta;CYCA3;2 protein, itscDNA was fused in frame to the 3

�

end of the cDNA encodingthe GFP, resulting in

GFP-Nicta;CYCA3;2

. The fusion constructunder the control of the constitutive

Cauliflower mosaic virus

35S promoter was introduced into both tobacco BY2 cells andArabidopsis plants. We obtained transgenic BY2 strains thatcontained only a small proportion (up to 15%) of cells showingdetectable GFP fluorescence. This finding possibly indicatesthat high levels of GFP-Nicta;CYCA3;2 are incompatible withBY2 cell growth. In cells expressing

GFP-Nicta;CYCA3;2

, thefluorescence was detected exclusively within the nucleus (Fig-ure 2A). In transgenic Arabidopsis plants expressing

GFP-Nicta;CYCA3;2

, fluorescence was detected primarily in cellswithin regions close to the hypocotyl root junction (Figures 2Cand 2D). The root tip did not show

GFP-Nicta;CYCA3;2

fluores-cence. This finding differs from what was seen in plants ex-pressing

H2B-CFP

under the control of the same promoter, inwhich fluorescent cells were distributed more uniformly in dif-ferent organs, including root tips (Figure 2B). It is likely thatmechanisms exist to downregulate the level of GFP-Nicta;CYCA3;2, particularly in cells of proliferating tissues (e.g., rootmeristems located in the tip).

In cells from different types of tissues, including the epider-mis and cortex of hypocotyls and roots, as well as in root hairs,GFP-Nicta;CYCA3;2 fluorescence again was located exclu-sively in the nuclei (Figures 2E and 2F). Strong fluorescent nu-clear bodies (Figure 2G), with a number varying from three tonine per nucleus in Arabidopsis, were observed in cells expressing

GFP-Nicta;CYCA3;2

, suggesting that the GFP-Nicta;CYCA3;2protein occupies particular nuclear territories and/or has spe-cific target regions in the nucleus. Distinct from H2B-CFP, whichwas incorporated in the nucleosome and showed chromatin fi-ber localization (Figures 2H and 2I), GFP-Nicta;CYCA3;2 was dif-fuse in the nucleoplasm (Figure 2G). GFP-Nicta;CYCA3;2 fluo-

Figure 2.

Localization of the GFP-Nicta;CYCA3;2 Fusion Protein inTransgenic Tobacco BY2 Cells and in Transgenic Arabidopsis Plants.

(A)

Epifluorescent and bright-field differential interference contrast (DIC)images of a tobacco BY2 cell expressing

GFP-Nicta;CYCA3;2

. Note thegreen fluorescence exclusively in the nucleus.

(B)

Overlay of confocal epifluorescent and DIC images of a root tip froman Arabidopsis plant expressing

H2B-CFP

. Note the green fluorescencein the nuclei of all of the imaged cells.

(C)

and

(D)

Epifluorescent images of the hypocotyl root

(C)

and the rootregions

(D)

of an Arabidopsis plant expressing

GFP-Nicta;CYCA3;2

.Note the green fluorescence in only some of the cells.

(E)

Overlay of confocal epifluorescent and DIC images of the Arabidop-sis root region expressing

GFP-Nicta;CYCA3;2

. Note the green fluores-cence in the nucleus of an epidermal cell.

(F)

Confocal epifluorescent, DIC, and overlay images of a root hair froman Arabidopsis plant expressing

GFP-Nicta;CYCA3;2

. Note the greenfluorescence exclusively in the nucleus and a few bright spots in nuclearbodies.

(G)

to

(I)

Projections of confocal image stacks.

(G)

and

(H)

Nuclei of the Arabidopsis root hairs expressing

GFP-Nicta;CYCA3;2

(G)

and

H2B-CFP

(H)

. Note the difference in the distribution ofnuclear bodies as well as the diffuse nucleoplasm and the chromatin fi-ber localization of GFP-Nicta;CYCA3;2 and H2B-CFP, respectively.

(I)

Nucleus of a root hair after propidium iodide staining. Note the redfluorescence of the stained DNA.Bars

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(F)

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(G)

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(I)

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2766 The Plant Cell

rescence was never detected in the nucleoli, whereas the GFP-Nicta;CYCA3;1 fusion protein has been shown to be localized inthe nucleus and nucleoli (Criqui et al., 2001), suggesting thatNicta;CYCA3;1 and Nicta;CYCA3;2 can have different nucleolarfunctions. We detected no GFP-Nicta;CYCA3;2 fluorescence inmetaphase cells, suggesting that the fusion protein may be de-graded at M-phase. Nicta;CYCA3;1 has been demonstrated tobe degraded at early M-phase (Genschik et al., 1998; Criqui etal., 2001).

Antisense

Nicta;CYCA3;2

Expression Induces Defects in Embryogenesis

To assess the consequences of the downregulation of

Nicta;CYCA3;2

on plant development, we cloned the

Nicta;CYCA3;2

cDNA in the antisense orientation behind the

TetO

promoter inthe pBinHyg-Tx vector (Gatz et al., 1992). Two types of to-bacco plants were used in this transformation:

TetR

homozy-gous plants, which were engineered to overexpress the TET re-pressor protein (Gatz et al., 1992), and

H4A748

homozygousplants, which were engineered to express the

�

-glucuronidase(GUS) reporter gene under the control of an Arabidopsis his-tone

H4

promoter (Lepetit et al., 1992). In the

TetR

background,transcriptional activity of the

TetO

promoter is repressed untilthe addition of tetracycline, whereas in the

H4A748

back-ground,

TetO

functions as a strong constitutive promoter.Transformation with antisense

Nicta;CYCA3;2

under the controlof the

TetO

promoter resulted in numerous transformants from

TetR

leaf discs. However, only a few transformants were regen-erated from

H4A748

leaf discs. The inhibition effect of theexpression of antisense

Nicta;CYCA3;2

on regeneration is dis-cussed below. Here, we present our studies on the transfor-mants obtained in the

H4A748

background.Of eight plants regenerated from different (parts of) leaf discs

and therefore originating from independent transformationevents, five plants were confirmed by PCR amplification for thepresence of antisense

Nicta;CYCA3;2

in their genomes. Theseplants (hereafter named lines

AS1

to

AS5

) grew without show-ing significant defects in morphology or development and wereallowed to set seeds by self-fertilization. Although

AS1

seedsgerminated with 100% efficiency and resulted in seedlingsidentical to the wild type,

AS2

,

AS3

,

AS4

, and

AS5

seedsshowed defects in germination and/or seedling growth, with 4,10, 46, and 36% of seeds, respectively (based on 500 to 600seeds plated for each line), blocked in germination and/orseedling growth. Figure 3A shows a germination plate of

AS4

seeds. The defective seeds can be classified into two groupsaccording to their germination capability. The first group, whichaccounted for a small percentage (

�

5%) of defective seeds, re-tained germination capability, but the seedlings exhibited se-vere distortions of morphology. The most affected seedlingslacked roots, hypocotyls, and shoot meristems, whereas theless affected seedlings lacked only shoot meristems (Figure3B). The second group of defective seeds was incapable ofgermination. Dissections of the seeds revealed that most embryoswere not properly formed (Figure 3C): embryonic roots, hypo-cotyls, and cotyledons could not be identified. Some embryos

appeared phenotypically normal but failed to express the reporterH4 promoter-GUS gene (Figure 3D), indicating defects in prolif-eration. Together, these observations indicate that Nicta;CYCA3;2is essential for embryo patterning and morphogenesis.

To verify the molecular effects of antisense Nicta;CYCA3;2,we analyzed the level of Nicta;CYCA3;2 mRNA and p13suc1 af-finity-purified CDK activity in germinated seedlings. Strong de-creases (greater than twofold) of both Nicta;CYCA3;2 mRNAand CDK activity (assayed using histone H1 as a substrate)were detected in the AS4 and AS5 lines (Figure 4). This findingis consistent with the strong defects of embryo formation ob-

Figure 3. Loss of Function of Nicta;CYCA3;2 Causes Defects in To-bacco Embryo and Seedling Formation.

Investigations were made using antisense Nicta;CYCA3;2 transgenicplants in the H4A748 background containing a GUS reporter gene un-der the control of a histone H4 promoter.(A) Germination plate of seeds from self-fertilization of the transgenicline AS4. Note the high number of seeds defective in germination. Thephotograph was taken 10 days after germination.(B) Normal (left) and defective (middle and right) seedlings of line AS4 at14 days after germination. Note the lack of a shoot meristem in the mid-dle seedling and the lack of roots, hypocotyls, and shoot meristems inthe seedling at right.(C) Normal (left) and defective (middle and right) embryos dissectedfrom the wild-type seed and defective AS4 seeds, respectively. Note thelack of formation of embryonic roots, hypocotyls, and cotyledons in thedefective embryos.(D) Histochemical detection of GUS activity in a wild-type embryo (mid-dle), an AS4 seedling (left), and an AS4 defective embryo (right). Notethe absence of GUS activity in the AS4 defective embryo.

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Nicta;CYCA3;2 and Plant Development 2767

served in these lines. Slight decreases in H4 mRNA levels alsowere noted in these lines (Figure 4). However, correlations be-tween the phenotype, the Nicta;CYCA3;2 mRNA level, and theCDK activity could not be established in the weakly affectedAS1, AS2, and AS3 lines. This discrepancy might reflect limita-tions of the detection sensitivity, but it also could be explainedby other, yet unknown mechanism(s) that compensate for thedownregulation of Nicta;CYCA3;2 mRNA level.

Antisense Nicta;CYCA3;2 Expression Inhibits Callus Regeneration

To further analyze the effects of the ectopic expression of anti-sense Nicta;CYCA3;2 on plant regeneration from leaf discs, wetested callus formation in vitro using inducible expression in theTetR background. Of 13 initially established transgenic linesfrom the transformation of TetR leaf discs, 3 lines were se-lected for this study for their high efficiency in reducing Nicta;CYCA3;2 mRNA levels upon tetracycline induction in seedlings.All three lines gave similar results, and we present here resultsobtained from one of them. In the absence of tetracycline, theantisense Nicta;CYCA3;2 leaf discs exhibited callus and shootregeneration similar to that in control TetO promoter-GUS leafdiscs (data not shown). However, in the presence of tetracy-cline, callus and shoot formation were inhibited significantly fromantisense Nicta;CYCA3;2 leaf discs compared with control leafdiscs (Figure 5A). The inhibition was even more pronouncedwhen the tests were performed in liquid medium (Figure 5B). Areduced number of cell divisions could be the cause of the ob-served inhibition, because cells of antisense Nicta;CYCA3;2calli were larger than those of control calli (Figures 5C and 5D),with cell areas of 28,176 � 3,158 (n � 67) and 17,333 � 2,639(n � 84) �m2, respectively.

Ectopic Expression of GFP-Nicta;CYCA3;2 Affects Arabidopsis Development

To investigate the effects of the overexpression of Nicta;CYCA3;2on plant development, we used the construct GFP-Nicta;CYCA3;2under the control of the constitutive 35S promoter. This allowsthe selection of transformants by detection of the fluorescenceof the fusion protein with a microscope. After difficulties in get-ting transgenic tobacco plants to overproduce the fusion pro-tein by leaf disc transformation, we chose to transform Arabi-dopsis plants by the floral-dip method (Clough and Bent, 1998),which does not require in vitro tissue regeneration. We screeneda large pool of seeds collected from the transformation of 16Arabidopsis plants. Twenty-one transformants were obtainedafter selection on kanamycin plates. Examination by microscopyrevealed that seven of them showed no detectable GFP fluo-rescence, and nine of them showed GFP-fluorescent cells (withthe number of cells ranging from 1 to �50) located in the hypo-cotyl and root in proximity to the junction of the two organs(Figure 2C). These transformants with no or weak GFP-Nicta;CYCA3;2 production grew and set seeds without a noticeablephenotype. A similar pattern of GFP fluorescence was ob-served in seedlings of the next generation from self-fertilizationof these plants. In seedlings of two lines showing a relatively

Figure 4. Nicta;CYCA3;2 Transcript Levels and CDK Kinase Activity inAntisense Transgenic Tobacco Plants.

Three-week-old seedlings of the wild type (lane C) and of the antisenseNicta;CYCA3;2 transgenic lines AS1 (lane 1), AS2 (lane 2), AS3 (lane 3),AS4 (lane 4), and AS5 (lane 5) in the H4A748 background were ana-lyzed.(A) An RNA gel blot was hybridized successively with Nicta;CYCA3;2,the histone H4, and the ribosomal 18S rRNA probes.(B) Histone H1 kinase activity was assayed on p13suc1-Sepharose affin-ity-purified CDK-cyclin complexes from equal amounts (200 �g) of totalplant proteins (H1 kinase). Equal loading of histone H1 (H1 protein) andthe immunodetection of PSTAIRE-containing CDK protein (@PSTAIRE)are shown as controls.(C) Quantitation of Nicta;CYCA3;2 and histone H4 transcript levels aswell as phosphorylated H1 levels was performed using a Phosphor-Imager (Molecular Dynamics, Sunnyvale, CA). Transcript levels werenormalized against the ribosomal 18S rRNA.

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high number of GFP-fluorescent cells, a slight inhibition of pri-mary root growth and accelerated secondary root growth werenoted early after germination (Figure 6B). However, no otherphenotype was observed in these first 16 transgenic lines.

By contrast, the other five transformants showed an in-creased number of GFP-fluorescent cells not only in roots andhypocotyls but also in leaves, indicating a high level of GFP-Nicta;CYCA3;2 protein in these lines. Striking developmentaleffects were observed in these lines. The two transformantsmaintained in vitro were incapable of developing root systems,their leaves curled over the abaxial surface, their inflorescencesceased elongation, and their flower buds died before opening,resulting in adult plants of miniature size (Figure 6A). The otherthree transformants were transferred to soil and grew undernormal greenhouse conditions. Two of them (hereafter namedOE1 and OE2) survived and showed a phenotype similar, butwith a lesser degree of severity, to that of the two in vitro–grown transformants, such as small and curled leaves, dwarfedstature, and sterility (Figures 6C and 6D). A bushy phenotypewas observed primarily because of a reduction of internodelength and an increased proliferation of lateral shoots. A highproliferation of lateral shoots was observed on flower stalks aswell as from the rosette (Figure 6E). Both the enhanced (pro-longed) proliferation state and the formation of new primordiacould be the cause of the high proliferation of lateral shoots.

A complete set of flower organs could be identified in OE1and OE2 plants. However, the stamens were too short to reachthe stigma for pollination. In addition, few pollen grains werefound in anthers, and these anthers did not dehisce to releasethe pollen. The fertility of the female part was examined by

hand pollination with pollen from wild-type plants. From eightsiliques resulting from hand pollination, we obtained 56 seeds,which is much lower than the 300 to 400 seeds normally ob-tained from wild-type plants, indicating defects in female fertil-ity. When these seeds were plated on germination medium, 13failed to germinate, indicating defects in embryo formation. Theother 43 seeds germinated, but only 1 showed resistance tokanamycin. This segregation ratio of 42 wild type to 14 (13 1)mutant is greater than the 1:1 ratio expected from the cross ofa heterozygous mutant plant with a wild-type plant, indicatingthat GFP-Nicta;CYCA3;2 production could interfere with femalegametogenesis. The kanamycin-resistant plant that resultedfrom the cross showed GFP fluorescence and had a phenotypesimilar to that of its maternal plant.

Cauline Leaves of Arabidopsis Plants Overexpressing GFP-Nicta;CYCA3;2 Display Reduced Endoreplication and Cell Size

To define the effects of gain of function at the cellular level, weanalyzed the cytology of cauline leaves of GFP-Nicta;CYCA3;2OE1 and OE2 plants. Cross-sections through the leaves re-vealed that the mutant leaves acquired the correct identity ofdifferent tissues: the vasculature, the adaxial and abaxial epi-dermis, and the mesophyll. Nevertheless, differences were ob-served when mutant and wild-type leaves were compared. Inthe wild-type leaf, the palisade cells that lie below the adaxialepidermis elongated and were arranged with their long axesperpendicular to the leaf surface, and the spongy mesophyllcells that lie between the palisade layer and the abaxial epider-

Figure 5. Antisense Expression of Nicta;CYCA3;2 Inhibits Callus Formation on Tobacco Leaf Discs.

Leaf discs from transgenic plants in the TetR background were analyzed for inducible expression with tetracycline.(A) Callus formation from leaf discs of control (left) and antisense Nicta;CYCA3;2 (right) cultured on solidified medium.(B) Callus formation from a representative leaf disc of control (left) and antisense Nicta;CYCA3;2 (right) cultured in liquid medium.(C) and (D) Representative calli cells of control (C) and antisense Nicta;CYCA3;2 (D) after staining with Lugol’s iodine-iodide solution. Bars � 30 �m.

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mis were smaller and more rounded (Figure 7A). In the mutantleaf, palisade cells were less elongated (Figure 7B), possibly in-dicating a lesser degree of differentiation from spongy meso-phyll cells. In addition, the mutant leaf contained one less layerof spongy mesophyll cells.

More striking effects were observed on both the adaxial andabaxial epidermis of leaves, which are characterized by tri-chomes, pavement cells, and stomata guard cells. Arabidopsisleaf trichomes are unicellular hairs that undergo several roundsof endoreplication during maturation, leading to a characteristicform of three branches (Melaragno et al., 1993). Although no sig-nificant effects on morphology or number of branches were ob-served, both nuclear DNA content and the cell size of trichomeswere reduced in the mutant (Figure 7D) compared with the wildtype (Figure 7C). The cell areas of mutant and wild-type tri-chomes were 5,669 � 1,411 (n � 52) and 10,638 � 3,913 (n �47) �m2, respectively. In addition, the large pavement epidermalcells with high DNA content observed in the wild-type leaf (Fig-ure 7E) were absent from the mutant leaf (Figure 7F). In the mu-tant leaf, the pavement cells were more uniform in size and thenumbers of pavement cells that separate guard cells from neigh-boring stomata were increased by a factor of �2. The ploidy lev-els of mutant and wild-type leaves were measured by flow cy-tometry. In wild-type leaves, cells presenting a DNA content of2C, 4C, 8C, or 16C were detected (Figure 7G). In mutant leaves,most of the cells had a DNA content of 2C and a small propor-tion of cells had a DNA content 4C, but cells with a DNA contentof �4C were barely detectable (Figure 7H). Together, these re-sults show that the ectopic overexpression of GFP-Nicta;CYCA3;2inhibits cell differentiation and endoreplication and modifies theproportion of cell size populations in leaves.

S-Phase–Specific Histone Gene Expression Is Upregulated in Arabidopsis Plants Overexpressing GFP-Nicta;CYCA3;2

To further determine the effects of the ectopic overexpression ofGFP-Nicta;CYCA3;2 on cell cycle events, we analyzed the expres-sion of cell cycle–specific genes in the OE1 and OE2 plants. Twogenes were chosen for this study: the histone H4A748 and the cy-clin Arath;CYCB1;1, which are expressed specifically duringS-phase and M-phase, respectively (Chaubet et al., 1996; Mengesand Murray, 2002). The transcript levels of these genes were com-pared between mutant and wild-type plants by semiquantitativereverse transcriptase–mediated PCR analysis using the constitu-tively expressed Actin2 gene as a standardization control. An up-regulation of the histone H4A748 by a factor of 4- to 10-fold wasfound in cauline leaves, stems, and flower buds of mutant plants(Figure 8). By contrast, the transcript level of Arath;CYCB1;1 wasnot affected in mutant plants. These results suggest a specific en-hancement of S-phase, through a premature entry and/or a de-layed exit, by the ectopic overexpression of GFP-Nicta;CYCA3;2.

GFP-Nicta;CYCA3;2 Overexpression in Arabidopsis Impairs Shoot and Root Regeneration in Tissue Culture

The failure to obtain transgenic tobacco plants overproducingGFP-Nicta;CYCA3;2 from leaf disc transformation suggestedan incompatible effect of the fusion protein on the regeneration

Figure 6. Phenotype of Arabidopsis Plants Overexpressing GFP-Nicta;CYCA3;2.

(A) A transformant with high expression of GFP-Nicta;CYCA3;2 at 60days after germination in vitro. The gradations at left are in 1-mm units.The arrowhead points to a flower stalk.(B) Two seedlings with moderate expression of GFP-Nicta;CYCA3;2(right) compared with two wild-type seedlings (left) at 7 days after ger-mination. The arrowheads point to newly forming lateral roots.(C) Transformant OE1 with high expression of GFP-Nicta;CYCA3;2(right) compared with a wild-type plant (left) at 60 and 45 days of growthin the greenhouse, respectively.(D) Close-up of a bushy (left) and a sterile (right) flower stalk of thetransformant OE1.(E) Transformant OE1 at 100 days of growth in the greenhouse. The ar-rowheads point to the highly proliferating lateral shoots from a flowerstalk.

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process. We tested this hypothesis by in vitro tissue culture ofleaf discs sampled from cauline leaves of wild-type and GFP-Nicta;CYCA3;2 overexpression Arabidopsis plants. Calli wereregenerated from leaf discs after culture on medium containinghigh concentrations of auxin and cytokinin (Figures 9A and 9B).At this step, no significant differences were noted betweencontrol and GFP-Nicta;CYCA3;2 samples. The regenerated callithen were assayed for shoot regeneration on medium contain-ing a decreased concentration of auxin. Abundant shoot regen-erations were observed on the control calli (Figures 9C and 9D)

but not on the GFP-Nicta;CYCA3;2 calli (Figures 9E and 9F).These late calli proliferated with unorganized masses of calli(Figure 9G), and the somatic embryo structure was not ob-served. Root regeneration also was assayed by transferring thecalli onto medium in the absence of cytokinin. The GFP-Nicta;CYCA3;2 calli proliferated with unorganized masses of calli(Figure 9H), whereas the control calli formed many root initials(Figure 9I). Together, these results demonstrate that GFP-Nicta;CYCA3;2 overexpression maintains cells in an undifferentiatedstatus that impairs shoot and root formation.

Figure 7. Effects of GFP-Nicta;CYCA3;2 Overexpression on Cell Division, Differentiation, and Endoreplication in Arabidopsis Leaves.

(A) and (B) Transverse sections through the central part of a mature cauline leaf of a control plant (A) and a GFP-Nicta;CYCA3;2 OE1 plant (B). Thepalisade (p) and spongy mesophyll (s) cells as well as the central vasculature (v) are indicated.(C) and (D) A wild-type trichome (C) and a GFP-Nicta;CYCA3;2 OE1 trichome (D). The insets show propidium iodide–stained nuclei.(E) and (F) A wild-type epidermis (E) and a GFP-Nicta;CYCA3;2 OE1 epidermis (F). The nuclei are heavily stained with 4�,6-diamidino-2-phenylindole(DAPI). The weaker stained cytoplasm also is visualized as a thin layer surrounding the cell as a result of large vacuoles. Note that large polygon-shaped pavement cells containing big spindle-form nuclei (one of them is marked by a magenta line) are present in the wild type but absent in GFP-Nicta;CYCA3;2 OE1.(G) and (H) Ploidy distribution of wild-type leaves (G) and GFP-Nicta;CYCA3;2 OE1 cauline leaves (H). The x and y axes display the relative DNA con-tent (log scale) and the number of DAPI-stained nuclei, respectively.Bars � 50 �m in (A) to (F) and 5 �m in the insets in (C) and (D).

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GFP-Nicta;CYCA3;2 Forms Active CDK Complexes in Arabidopsis

To verify if GFP-Nicta;CYCA3;2 forms active CDK complexes inArabidopsis, we combined affinity and immunoprecipitation as-says with protein gel blot analyses. Protein gel blotting withanti-GFP antibody revealed a band of the expected size for thefusion protein present specifically in samples of flower buds ofthe OE1 and OE2 plants and the GFP-Nicta;CYCA3;2 calli butabsent from the wild-type controls (Figure 10A, top). Consis-tently, increases of p13suc1 affinity-purified CDK activity weredetected in GFP-Nicta;CYCA3;2 samples (Figure 10A). The fol-low-up pulldown experiments were performed using undiffer-entiated calli because of their high level of GFP-Nicta;CYCA3;2production and the good quantity of materials. When the samefractions of p13suc1 affinity-purified CDK were subjected to ananalysis for histone H1 kinase activity as well as to protein gelblot analyses with anti-PSTAIRE and anti-GFP antibodies (Fig-ure 10B), we observed a repeated increase of kinase activ-ity and the presence of the fusion protein specifically in theGFP-Nicta;CYCA3;2 sample but a relatively equal amount of

PSTAIRE-containing CDK in the control and GFP-Nicta;CYCA3;2samples. Immunoprecipitation with the anti-GFP antibody re-sulted in H1 kinase activity present specifically in the GFP-Nicta;CYCA3;2 sample but absent from the control sample(Figure 10C). An endogenous protein was phosphorylated andfound nonspecifically in immunoprecipitates from both GFP-Nicta;CYCA3;2 and control samples. Both PSTAIRE-containingCDK and GFP-Nicta;CYCA3;2 proteins were detected andpresent specifically in immunoprecipitate from the GFP-Nicta;CYCA3;2 sample but not in that from the control sample (Figure10C). Together, these results demonstrate that GFP-Nicta;CYCA3;2 forms active kinase complexes with PSTAIRE-con-taining CDK proteins in Arabidopsis and that CDK proteins arepresent in nonlimiting, excess amounts.

DISCUSSION

Is Nicta;CYCA3;2 a Functional Analog of Animal cyclin E?

The G1/S transition constitutes an important checkpoint of themitotic cell cycle in all eukaryotes, in which the cells orientateto continue or to stop dividing. In animals, this transition pro-cess is coordinated by CDK-cyclin kinase complexes (Sherr,1994; Coverley et al., 2002). Reentering the cell cycle from qui-escence (G0 to G1 transition) involves cyclin D–CDK4/CDK6complexes that are activated by developmental and nutritionalcues. The cyclin E–CDK2 complex is involved in the mid G1-to-Stransition by stimulating the assembly of prereplication com-plexes for DNA synthesis. The cyclin A–CDK2 complex pro-motes S-phase progression by activating DNA synthesis on thereplication complexes that are already assembled and ensuresone round of replication by inhibiting the assembly of new pre-replication complexes (Coverley et al., 2002). In the model plantArabidopsis, homology-based sequence analyses of the entiregenome had identified and annotated 10 CYCD and 10 CYCAgenes (Vandepoele et al., 2002). Strikingly, however, no ho-molog of cyclin E was identified from the Arabidopsis genomeor reported from any other plant species. It is possible thatsome CYCD or CYCA genes might fulfill functions analogous tothose of cyclin E in plants. Knockout of Arath;CYCD3;2 has noobservable phenotype because of possible redundant func-tions by other CYCDs (Swaminathan et al., 2000). Functionalanalyses by ectopic overexpression and molecular character-izations revealed that Arabidopsis CYCD3;1 and CYCD2;1 andtobacco CYCD3;3 exhibit characteristics of animal cyclin D(Riou-Khamlichi et al., 1999; Cockcroft et al., 2000; Nakagamiet al., 2002; Dewitte et al., 2003, and references therein). Thefunctions of other CYCDs are currently uncharacterized.

Some molecular data indicate that different CYCAs couldhave different functions in plants. First, CYCAs can be classi-fied into three distinct phylogenic groups, and representativemember(s) for each group can be found in single plant species(Chaubet-Gigot, 2000; Vandepoele et al., 2002), suggestingthat different group members are evolutionarily conserved toperform specific functions. Second, different CYCAs of to-bacco show distinct expression profiles during the cell cycle(Reichheld et al., 1996), suggesting that different CYCAs per-

Figure 8. Expression Analysis of Cell Cycle Genes in GFP-Nicta;CYCA3;2–Overexpressing Arabidopsis Plants.

The S-phase–specific histone H4 and the M-phase–specific cyclin Arath;CYCB1;1 were analyzed by semiquantitative reverse transcriptase–mediated PCR using Actin2 as a reference gene.(A) and (B) Samples from flower buds of wild-type plants (A) and GFP-Nicta;CYCA3;2 OE1 plants (B).(C) Samples from cauline leaves of wild-type (left lane) and GFP-Nicta;CYCA3;2 OE1 (right lane) plants.(D) Samples from stems of wild-type (left lane) and GFP-Nicta;CYCA3;2OE1 (right lane) plants.The numbers above the lanes indicate the number of PCR cycles. Theasterisks indicate that fewer PCR cycles are required in (B) than in (A)for an equal production of histone H4, indicating an increase of approx-imately fourfold in the H4 transcript level in GFP-Nicta;CYCA3;2 OE1flower buds.

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form sequentially required specific functions. Third, differentCYCA proteins are found in particular cellular compartments.The maize A1-type cyclin Zeama;CYCA1;1 is localized in thecytoplasm in interphase, concentrated around the nucleus inprophase, and associates with the preprophase band, mitoticspindle, and phragmoplast during mitosis (Mews et al., 1997),whereas Medsa;CYCA2;2 is localized in the nucleus (Roudier etal., 2000). Nicta;CYCA3;1 (Criqui et al., 2001) and Nicta;CYCA3;2 (this work) also are both localized in the nucleus, butthey differ by their presence and absence, respectively, fromthe nucleoli. These data suggest that different CYCA proteinsmay perform compartment-specialized functions. Fourth, dif-ferent CYCA proteins might be regulated differently by proteol-ysis. Nicta;CYCA3;1 is degraded early after cells enter mitosis(Genschik et al., 1998; Criqui et al., 2001), whereas Zeama;CYCA1;1 is relatively stable, as shown by the fact that it can bedetected throughout mitosis (Mews et al., 1997).

In spite of this accumulation of molecular data, a direct func-tional demonstration of plant CYCAs had long been missing.

Recently, Roudier et al. (2003) demonstrated that antisense ex-pression of Medsa;CYCA2;2 in alfalfa inhibits shoot and rootdevelopment in tissue culture. We had shown previously thattransient induction of the ectopic overexpression of Nicta;CYCA3;2 at local regions of the tobacco shoot apical meristemand leaf primordia can induce cell divisions (Wyrzykowska etal., 2002). However, when the induction was performed at thewhole-plant level, significant effects on plant growth and devel-opment were not detected, in spite of highly increased levels ofNicta;CYCA3;2 mRNA (Wyrzykowska et al., 2002; our unpub-lished results). These previous results suggest that the role ofNicta;CYCA3;2 in cell division is dependent on the develop-mental context (Wyrzykowska et al., 2002) and that mecha-nism(s) exist to discriminate (or compensate) high levels ofNicta;CYCA3;2 mRNA. Consistent with this last assumption,Roudier et al. (2003) reported that overexpression of Medsa;CYCA2;2 does not result in the overproduction of Medsa;CYCA2;2 and has no effect on plant development. Here, wefused Nicta;CYCA3;2 to GFP, which allows the visualization of

Figure 9. Effects of GFP-Nicta;CYCA3;2 Overexpression on Callus Formation, and Subsequent Shoot and Root Regeneration from Arabidopsis LeafExplants.

(A) and (B) Callus regeneration from leaf explants in the presence of high concentrations of auxin and cytokinin. Samples from wild-type and GFP-Nicta;CYCA3;2 OE1 cauline leaves gave similar results. Only calli from GFP-Nicta;CYCA3;2 OE1 samples are shown.(C) and (D) Shoot regeneration from wild-type calli in the presence of a low concentration of auxin and a high concentration of cytokinin.(E) and (F) Absence of shoot regeneration from GFP-Nicta;CYCA3;2 calli under the same conditions as described for (C) and (D).(G) Section of a proliferating region of the callus in (F) showing an unorganized structure: the shoot, root, and somatic embryo structure could not beobserved.(H) and (I) Representatives of GFP-Nicta;CYCA3;2 and wild-type calli cultured in the absence of cytokinin showing the absence (H) and presence (I)of root regeneration, respectively.Bars � 100 mm in (B), (D), (F), (H), and (I) and 50 �m in (G).

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expression at the protein level. We demonstrated that GFP-Nicta;CYCA3;2 forms active CDK complexes in Arabidopsisand that its overproduction affects plant development dramati-cally.

Several of our observations indicate similarities betweenNicta;CYCA3;2 function in plants and cyclin E function in ani-mals. First, our antisense experiments demonstrated that Nicta;

CYCA3;2 is involved in the reentry into cell division by differen-tiated tissues of leaves in which most of the cells are arrested inG1 (G0) phase (our cytometric data not shown). This is similarto the requirement of cyclin E in the G1/S transition in animals(Knoblich et al., 1994). Second, the gain of function by the ec-topic expression of GFP-Nicta;CYCA3;2 inhibited cell differenti-ation and enhanced the expression of the S phase–specific his-tone gene. These data are consistent with the ectopic effects ofcyclin E in inducing the premature entry into S-phase in animals(Knoblich et al., 1994; Richardson et al., 1995; Lukas et al.,1997). Third, contrary to its S-phase function, cyclin E activityneeds to be reduced to allow endoreplication in Drosophila em-bryogenesis (Weiss et al., 1998; Edgar and Orr-Weaver, 2001).Similarly, ectopic overproduction of GFP-Nicta;CYCA3;2 inhib-ited endoreplication in Arabidopsis leaves. Together with thefact that the expression of Nicta;CYCA3;2 is activated particu-larly early at the G1/S transition (Reichheld et al., 1996), thesesimilarities prompted us to propose that Nicta;CYCA3;2 mayperform a function analogous to that of animal cyclin E in thecontrol of plant cell division and differentiation. Future experi-ments will be necessary to characterize Nicta;CYCA3;2 at themolecular level for its analogous cyclin E and/or cyclin A func-tion, particularly its relationship with the activation of the RBR/E2F pathway.

Cell Cycle and Embryo Formation

In contrast to the well-documented importance of cyclins in an-imal embryogenesis, the functions of cyclins in plant embryoformation have not been reported. Our results from antisensetransgenic tobacco plants show that Nicta;CYCA3;2 is requiredfor embryo formation and that its loss of function results in apanoply of embryonic phenotypes. The most severe pheno-types of the embryos, as dissected from germination-defectiveseeds, were the lack of morphologically recognizable cotyle-dons and embryonic roots (Figure 3C). Because the apical-basal axis is visible in these embryos, we believe that cotyle-dons and root primordia are initiated but that the cell divisionsnecessary for their growth are stopped at early stages. Distor-tions in embryo pattern formation also were shown by seed-lings with abnormal phenotypes. Seedlings that lack the shootmeristem alone or together with the hypocotyl and the root(Figure 3B) were observed, indicating severe defects in the for-mation of the corresponding embryonic organs. A range ofseedling phenotypes indicating distortions in embryo patternformation also have been reported in transgenic Arabidopsisplants overexpressing a dominant-negative mutant of the CDKA;1gene under the control of the 2S2 albumin promoter (Hemerlyet al., 2000). The PSTAIRE-containing CDKA;1 protein is presentconstitutively during the cell cycle and can interact with allthree (A, B, and D) types of plant cyclins (Stals and Inzé, 2001).Therefore, it is reasonable that the effects observed for thedominant-negative mutant CDKA;1 might be (partially) attribut-able to interactions of the protein with an Arabidopsis analog ofNicta;CYCA3;2. Four A3-type cyclins are present in the Arabi-dopsis genome (Vandepoele et al., 2002), but their functionsare currently uncharacterized.

Figure 10. GFP-Nicta;CYCA3;2 Protein Accumulation and Its Associ-ated CDK Activity in Arabidopsis.

(A) Total protein extracts from flower buds of wild-type (lane 1), GFP-Nicta;CYCA3;2 OE1 (lane 2), and GFP-Nicta;CYCA3;2 OE2 (lane 3)plants as well as from wild-type (lane 4) and GFP-Nicta;CYCA3;2 (lane5) calli were analyzed for the presence of GFP-Nicta;CYCA3;2 proteinand histone H1 kinase activity. GFP-Nicta;CYCA3;2 was detected byprotein gel blot analysis using a rabbit polyclonal anti-GFP antibody(@GFP). H1 kinase activity was assayed on p13suc1-Sepharose affinity-purified CDK-cyclin complexes from 100 �g of total proteins (H1 kinase).Equal loading of histone H1 (H1 protein) and immunodetection ofPSTAIRE-containing CDK (@PSTAIRE) are shown as controls.(B) p13suc1-Sepharose affinity-purified fractions from 200 �g of totalproteins of wild-type (lane 4) and GFP-Nicta;CYCA3;2 (lane 5) calli wereassayed simultaneously for H1 kinase activity (H1 kinase), the presenceof PSTAIRE-containing CDK (@PSTAIRE), and GFP-Nicta;CYCA3;2 pro-tein (@GFP).(C) Immunoprecipitates from the rabbit polyclonal anti-GFP antibodyfrom 300 �g of wild-type (lane 4) and GFP-Nicta;CYCA3;2 (lane 5) calliwere assayed simultaneously for H1 kinase activity (H1 kinase), thepresence of PSTAIRE-containing CDK (@PSTAIRE), and GFP-Nicta;CYCA3;2 protein (@GFP).Open arrowheads point to the positions of PSTAIRE-containing CDKproteins, closed arrows point to the positions of the GFP-Nicta;CYCA3;2fusion proteins, and the asterisk indicates an unknown protein that isphosphorylated and present in both wild-type and GFP-Nicta;CYCA3;2immunoprecipitates.

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Endoreplication, Cell Expansion, and Postembryonic Development

Our gain-of-function experiments with transgenic Arabidopsisdemonstrate that the ectopic overexpression of GFP-Nicta;CYCA3;2 results in a profound perturbation of postembryonicdevelopment. In Arabidopsis leaf epidermal cells, the arrest ofcell division is followed by the onset of endoreplication, leadingto increased nuclear DNA content and cell size (Traas et al.,1998). Plants overexpressing GFP-Nicta;CYCA3;2 have a dra-matically decreased level of endoreplication in leaves, indicat-ing an inhibition of cell differentiation. This reduces organ andplant size and modifies the morphology of the plant, revealingan important function of endoreplication and the correspondingcell expansion in pattern formation and development. These re-sults are in contrast to those obtained with transgenic plantsoverexpressing the negative-dominant CDKs, Arath;CYCB1;1,or Arath;CYCD2;1, in which transgene expression modified therate of cell division but not cell differentiation or plant morphol-ogy (Hemerly et al., 1995, Doerner et al., 1996, Cockcroft et al.,2000; Porceddu et al., 2001). Our results also differ from thoseobtained with transgenic Arabidopsis overexpressing ICK1/KRP1/KIS1, in which the reduced cell division and endoreplica-tion were compromised by increased cell size, leading to plantswith a different phenotype (Wang et al., 2000; De Veylder et al.,2001; Jasinski et al., 2002). Interestingly, overexpression of Ar-ath;CYCD3;1 and E2Fa-DPa both induced hyperplasia but af-fected endoreplication oppositely, decreasing and increasing it,respectively, leading in both cases to profound defects in plantdevelopment (De Veylder et al., 2002; Dewitte et al., 2003;Kosugi and Ohashi, 2003).

Endoreplication consists of one or several rounds of DNAsynthesis without mitosis, which requires exit from the mitoticcycle and transformation of the cell cycle to the endocycleby inhibiting the G2-to-M transition. Overexpression of Arath;CYCB1;2 in trichomes of Arabidopsis induced ectopic cell divi-sion, resulting in a reduced level of polyploidy of individual nu-clei (Schnittger et al., 2002b). Consistent with the requirementfor the inactivation of mitosis-promoting factors in endoreplica-tion, downregulation of the fizzy-related ccs52 gene, which isinvolved in the destruction of mitotic cyclins, resulted in an in-creased level of polyploidy in alfalfa plants (Cebolla et al.,1999). The requirement of S-phase activity in plant endorepli-cation is suggested by the finding that polyploid maize en-dosperm contains high S-phase CDK activities (Grafi andLarkins, 1995).

However, the role of G1/S regulators in endoreplication is notunderstood completely. On the one hand, overexpression ofE2Fa-DPa activates the expression of S-phase genes and in-duces endoreplication in Arabidopsis and tobacco (De Veylderet al., 2002; Kosugi and Ohashi, 2003). On the other hand, Ara-bidopsis plants overexpressing Arath;CYCD3;1, an activator ofthe RBR/E2F-DP pathway, exhibited a reduced level of endo-replication (Dewitte et al., 2003). Schnittger et al. (2002a)demonstrated that overexpression of Arath;CYCD3;1 in tri-chomes of Arabidopsis induces not only DNA replication butalso ectopic cell division, suggesting that Arath;CYCD3;1 canpromote mitosis in addition to the G1/S transition. Distinct from

Arath;CYCD3;1, overexpression of GFP-Nicta;CYCA3;2 did notinduce extra cell divisions (hyperplasia). This finding promptedus to propose that a failure to exit the mitotic cycle and/or toinitiate DNA replication of the endocycle could cause the inhibi-tion of endoreplication by GFP-Nicta;CYCA3;2. Consistent witha role of D3- and A3-type cyclins in the control of endoreplica-tion, transcripts of Lyces;CYCD3;1 and Lyces;CYCA3;1 but notof Lyces;CYCA1;1, Lyces;CYCA2;1, Lyces;CYCB1;1, and Ly-ces;CYCB2;1 were detected in endoreplication gel tissue of to-mato (Joubes et al., 2000). Expression analysis of Nicta;CYCA3;2 in endoreplication cells, at both the transcript andprotein levels, might add to our understanding of its role in en-doreplication.

Cell Dedifferentiation, Callus Formation, and Organogenesis

When explanted into culture under certain exogenous stimuli,many plant tissues dedifferentiate, proliferate to form calli, andundergo organogenesis. Hemerly et al. (1993) found that theexpression of PSTAIRE-containing CDK correlates with thecompetence of cell division but that the release of other con-trols seems to be necessary for cell division to occur. Over-expression of Arath;CYCD3;1 can overcome the cytokinin re-quirement of callus formation in transgenic Arabidopsis leafexplants (Riou-Khamlichi et al., 1999), indicating its importantrole in the release of dedifferentiation and subsequent cell divi-sion. The Arabidopsis calli overexpressing Arath;CYCD3;1 aredefective in shoot regeneration (Riou-Khamlichi et al., 1999).We found that the ectopic overproduction of GFP-Nicta;CYCA3;2 impairs shoot as well as root regeneration from trans-genic Arabidopsis calli. It is likely that organogenesis requires adecreased level of CDK activity. This assumption is supportedby the findings that ectopic overexpression of a rice CDK-acti-vating kinase in tobacco converted root organogenesis to dis-organized callus proliferation (Yamaguchi et al., 2003) and thatthe ectopic overexpression of Medsa;CYCB2;2 in tobacco in-hibited root development from tissue-cultured leaf explants(Weingartner et al., 2003). Medsa;CYCB2;2 has been docu-mented to have molecular properties similar to those of animalcyclin A (Weingartner et al., 2003).

In addition to the overexpression experiments, we found thatantisense expression of Nicta;CYCA3;2 inhibited callus forma-tion and shoot regeneration from transgenic tobacco leaf ex-plants, indicating its requirement in cell division. However, itwas difficult to monitor the change of Nicta;CYCA3;2 transcriptlevel during the early phase of callus formation because the ex-pression of Nicta;CYCA3;2 was extremely low in leaves and thededifferentiation process leading to callus formation occurredin a small number of differentiated cells within the vascularbundle. Nevertheless, it is interesting that antisense expressionof Medsa;CYCA2;2 did not interfere with callus and somaticembryo formation from transgenic alfalfa leaf explants, whereassubsequent shoot and root development was inhibited (Roudieret al., 2003). It is possible that different cyclins play differentroles in dedifferentiation, proliferation, and redifferentiation. Itwas reported that different cyclin genes respond differently to

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exogenous stimuli. Medsa;CYCA2;2 expression is upregulatedby auxin (Roudier et al., 2003), Arath;CYCD3;1 expression isactivated by cytokinin (Riou-Khamlichi et al., 1999), and Arath;CYCD2;1 and Arath;CYCD4;1 expression is induced by su-crose (De Veylder et al., 1999; Riou-Khamlichi et al., 2000). Pro-moter isolation and characterization of Nicta;CYCA3;2 expres-sion under different physiological conditions will help increaseour understanding of its role in callus formation and organo-genesis. Further characterization of cell cycle genes in organo-genesis and embryogenesis will be required to understand theregulatory mechanisms of plant development. The observationthat modified expression of cell cycle regulators interferes withplant growth, morphogenesis, and/or in vitro regenerationcould have an important impact on biotechnology.

METHODS

Plant Materials

Tobacco (Nicotiana tabacum) and Arabidopsis thaliana (Columbia eco-type) plants were grown under greenhouse conditions. Tobacco BY2 (cvBright Yellow 2) cell suspension was maintained by weekly subculture asdescribed (Nagata et al., 1992).

Nicta;CYCA3;2 Constructs

To construct the GFP-Nicta;CYCA3;2 fusion, the entire coding region ofNicta;CYCA3;2 cDNA was amplified by PCR using the primers 5�-GCG-GATCCATGAAGAAGCAAGAGAAAGAGGC-3� and 5�-CCTCTAGATTAC-AATTGTCTCATATCTTC-3�. The PCR product was cloned into the pKS-GFP vector using BamHI and XbaI restriction sites, resulting in thein-frame fusion of GFP and Nicta;CYCA3;2. The GFP-Nicta;CYCA3;2 fu-sion fragment was isolated by a first digestion with XhoI, blunt-endingwith Klenow, and a second digestion with SacI. It was introduced intothe binary vector pBI121 (Clontech Laboratories, Palo Alto, CA) that hadbeen opened previously by a first digestion with BamHI, blunt-endingwith Klenow, and a second digestion with SacI, resulting in GFP-Nicta;CYCA3;2 under the control of the 35S promoter and the NOPALINESYNTHASE terminator.

The antisense Nicta;CYCA3;2 construct was made by PCR amplifica-tion of Nicta;CYCA3;2 cDNA using the primers 5�-ATTAATGGTACC-GTCGACTCTTGCTTTTCTTTGTTAGTTC-3� and 5�-ATTAATGGTACC-TCTAGATTGTATGTCCAAGCAGAAGCC-3� and by insertion of the PCRfragment behind the TetO promoter of the binary vector pBinHyg-Tx(Gatz et al., 1992) using the KpnI and SalI restriction sites.

Plant Transformation and Tissue Culture

Tobacco plant transformation from leaf explants and the establishmentof transgenic BY2 cell lines were as described previously (Shen, 2001).Callus regeneration from transgenic tobacco leaf explants was assayedunder the same conditions used in transformation in the presence of0.54 �M naphthylacetic acid (NAA) and 6.5 �M benzylaminopurine(BAP). Transgenic Arabidopsis plants were obtained by Agrobacteriumtumefaciens–mediated transformation using the floral-dip method (Cloughand Bent, 1998). Arabidopsis calli were regenerated from cauline leaf ex-plants in the presence of 2.3 �M 2,4-D, 11.4 �M indoleacetic acid, and3.2 �M BAP. Shoot regeneration from Arabidopsis calli was assayed inthe presence of 0.54 �M NAA and 3.2 �M BAP, and root regenerationwas assayed in the presence of 0.54 �M NAA alone.

RNA Analysis

Total RNA was prepared from plant tissues using the Trizol reagent kit(Gibco BRL). For RNA gel blot analysis, aliquots of 25 �g of RNA wereanalyzed by electrophoresis on formaldehyde-agarose gels, blotted ontoHybond-N nylon membranes (Amersham), and hybridized successivelywith different 32P-labeled probes under standard high-stringency condi-tions (Sambrook et al., 1989). The probes were as described previously(Reichheld et al., 1996; Chabouté et al., 1998). Reverse transcriptase–mediated PCR analyses were performed using the Avantage-2 kitaccording to the manufacturer’s instructions (Clontech Laboratories).The gene-specific primers used in reverse transcriptase–mediated PCRwere 5�-CGAGATAAACTAAATCTTCGC-3� and 5�-AAACTCTAATTAACC-ACCGA-3� for H4A748, 5�-AAGCTTCCATTGCAGACGA-3� and 5�-AGC-AGATTCAGTTCCGGTC-3� for Arath;CYCB1;1, and 5�-AAGTCATAA-CCATCGGAGCTG-3� and 5�-ACCAGATAAGACAAGACACAC-3� for Actin2.

Histochemical GUS Activity Assay

Seedlings and embryos dissected from defective seeds of Nicta;CYCA3;2AS4 at 8 days after germination, as well as embryos dissected from wild-type seeds at 2 days after germination, were incubated in 50 mM sodiumphosphate buffer, pH 7, containing 0.2 mM 5-bromo-4-chloro-3-indolyl-�-D-glucuronide, 0.5 mM potassium ferricyanide, 0.5 mM potassium fer-rocyanide, and 0.5% (v/v) Triton X-100 at 37C overnight. Photographswere taken of representative seedlings and embryos with a binocular mi-croscope.

Immunoblotting, Immunoprecipitation, p13suc1-Sepharose Affinity Binding, and Histone H1 Kinase Assays

Immunoprecipitation, p13suc1-Sepharose affinity binding, histone H1 ki-nase reaction, and protein gel blotting were performed as described pre-viously (Criqui et al., 2001). The affinity-purified anti-PSTAIRE rabbitpolyclonal antibody was purchased from Santa Cruz Biotechnology(Santa Cruz, CA) and used at a 4500-fold dilution in protein gel blot anal-yses. The affinity-purified anti-GFP rabbit polyclonal antibody was pur-chased from Molecular Probes (Leiden, The Netherlands) and used at a2500-fold dilution in protein gel blot analyses and at a 150-fold dilution inimmunoprecipitation. The mouse anti-GFP monoclonal antibody waspurchased from Roche Applied Science (Meylan, France) and used at a2500-fold dilution in protein gel blot detection of the GFP fusion proteinfrom the immpunoprecipitates.

Histology and Microscopy

Microscopic examination was performed as described previously (Shen,2001). For histological analysis, samples were fixed in 10% formalin, 5%acetic acid, and 50% ethanol, embedded in Paraplast (Oxford Labware,St. Louis, MO), and sectioned at 10 �m.

Flow Cytometry

After removal of the leaf petioles, leaf blades were chopped with a razorblade in Galbraith’s buffer (Galbraith et al., 1983). After filtration over a30-�m mesh, the nuclei were stained with 4�,6-diamidino-2-phenylin-dole and analyzed with a UV flow cytometer (EPS Elite; Beckman-Coulter).

Upon request, materials integral to the findings presented in this pub-lication will be made available in a timely manner to all investigators onsimilar terms for noncommercial research purposes. To obtain materials,please contact W.-H. Shen, [email protected].

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ACKNOWLEDGMENTS

This article is dedicated to the memory of Claude Gigot (deceased in1997), the leader for the initiation of this project. We thank IsabelleBisson and Roberte Bronner for help with plasmid construction and his-tology, respectively. We are grateful to Chris Bowler and Fabio Formiggini(Laboratory of Molecular Plant Biology, Stazione Zoologica, Naples,Italy) for the H2B-CFP plasmid. We thank Thomas Potuschak for criti-cally reading the manuscript. Y.Y. is supported by a fellowship from theAssociation Franco-Chinoise pour la Recherche Scientifique et Tech-nique. The InterInstitut confocal microscopy plate form was cofinancedby the Centre National de la Recherche Scientifique, the Université LouisPasteur, the Région Alsace, and the Association pour la Recherche surle Cancer.

Received August 4, 2003; accepted September 24, 2003.

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The Tobacco A-Type Cyclin, Nicta;CYCA3;2, at the Nexus of Cell Division and Differentiation