Gene, 105 (1991) 159-165

0 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/$03.50 159

GENE 06018

Identification of two cell-cycle-controlling cdc2 gene homologs in Arabidopsis thaliana

(Recombinant DNA; cDNA; higher plant; nucleotide sequence; gene expression; Schizosaccharomyces pombe)

Takashi Hirayama”, Yoshiro Imajuku”, Toyoaki Anai b, Minami Matsui b and Atsuhiro Oka”

“ Institute for Chemical Research, Kyoto University, Uji, Kyoto (Japan); and b Institute of Gerontology, Nippon Medical School, Nakahara-Ku, Kawasaki, Kanagawa (Japan) Tel. (81)044-733-5230

Received by H. Yoshikawa: 8 April 1991 Revised/Accepted: 25 May/9 June 1991 Received at publishers: 27 June 1991

SUMMARY

The cdc2 gene product (~34’~‘~) has been thought to play a central role in control of the mitotic cell cycle of yeasts and animals. To approach an understanding of the cell-cycle-control system in higher plants, we isolated, from an Arabidopsis

thaliana cDNA library, two clones (CDC2a and CDC2b) similar to the Schizosaccharomyces pombe cdc2 gene. Genomic Southern-blot analysis with the CDC2a and CDC2b cDNA probes suggested that the A. thaliana genome contains several additional cdc2-like genes, which together with the CDC2a and CDC2b genes may constitute a CDC2 gene family. The CDC2a

cDNA expressed in SC. pombe corrected the elongated morphology, caused by the temperature-sensitive cdc2-33 mutation, to the normal shapes, indicating that the A. thahana CDC2a gene product resembles SC. pombe ~34”~“~ functionally as well as structurally. These results support the view that the cell cycle of higher plants is controlled by an analogue of a

P34cd’2 -centered regulatory system like that of yeasts and animals.

INTRODUCTION

Cell division in higher plants is generally restricted to meristems. Little is known about plant mitotic regulation beyond the conditional control points during the cell cycle which were posed by Van? Hof (1966). On the other hand, control of the cell division cycle in yeasts has been exten- sively studied using both genetic and biochemical approaches. In the fission yeast Sc.pombe, the cdc2 gene product (~34’~“) appears to have a principal role in cell-

Correspondence 10: Dr. A. Oka, Institute for Chemical Research, Kyoto

University, Uji, Kyoto 611 (Japan)

Tel. (81)0774-32-8336; Fax (81)0774-33-1247.

Abbreviations: A., Arabidopsis; aa, amino acid(s); cDNA, DNA comple-

mentary to mRNA; kb, kilobase( nt, nucleotide(s); oligo, oligodeoxyri-

bonucleotide; ORF, open reading frame; p34“““, 34-kDa protein en-

coded by cdc2; Sa., Saccharomyces; SC., Schizosaccharomyces; SDS,

sodium dodecyl sulfate; SSC, 150 mM NaCl/lS mM Na, . citrate pH 7.2;

[ 1. denotes plasmid-carrier state.

cycle control by modulating its protein serine/threonine kinase activity (for reviews, see Lewin, 1990; Nurse, 1990). Defects in the cdc2 gene lead to arrest (i) at the ‘start’ point in late Gl for commitment to the mitotic cell cycle and (ii) at the G2-M transition (Nurse and Bissett, 1981). In the budding yeast Sa. cerevisiae, the protein encoded by the homologous gene CDC28 is required at the corresponding dual times in the cell cycle (Reed and Wittenberg, 1990). These two control points are compatible with those of the higher plant cell cycle hypothesized above.

Homologs of the cdc2/CDC28 genes have been found in a variety of animals (as reviewed in Norbury and Nurse, 1989). Furthermore, it has been shown with both Xenopus

laevis (Dunphy et al., 1988; Gautier et al., 1988) and star- fish (Labbe et al., 1989) that ~34’~~~ is a component of the M-phase promoting factor. The activity of these animal p34’d’2 kinases varies during the cell cycle and peaks in M-phase like that of yeast ~34’~” (Arion et al., 1988; Draetta and Beach, 1988; Labbe et a1.,1988), being re- gulated both by the level of its own phosphorylation

160

(Draetta and Beach, 1988; Simanis and Nurse, 1986) and by interaction with other proteins (Brizuela et al., 1987; Draetta et al., 1987; Mendenhall et al., 1987; Dunphy et al., 1988; Lohka et al., 1988).

The omnipresence of c&2 homologs together with the similarity of the cell-cycle-control points in plants and yeasts implies that higher plants probably contain a cdc2 homolog conducting their cell division cycles. In fact,

P34=jC2 -like proteins have been found in several plants, and a polymerase-chain-reaction fragment corresponding to a putative cdc2 gene has been isolated from garden peas (Feiler and Jacobs, 1990). As a further step toward under- standing the control systems for higher plant cell cycles, we now show that a flowering plant. A. thaliana, seems to

contain several cdc24ike genes, based on isolation of two cdc2 homologs (CDC2a and CDC2b) from a cDNA library and genomic Southern-blot analysis. In addition, functional similarity between the A. thaliana CDC2a and SC. pombe cdc2 genes is demonstrated by complementation test.

RESULTS AND DISCUSSION

(a) Isolation and characterization of an Arab&p%

thaliaaa c&2 homolog

Plaques from an A. thaliana (Columbia ecotype) cDNA library constructed with Igt 10 vector were screened under moderate-stringency hybridization conditions with the

LARAF61PVRTFTHEVVTLWYRAPE1LL6SHH~STPVDIVSV6CIFAEK~~198~

T~A..&.TCA..T. TG6NWZTAACTTCTCT’TFAT~TCT~TMA~ S Q K P L F P 6 D S E I D Q L Ixypy. I F R I II 6 T P Y E D T W R 6&T S L P D Y K S A F P K W K P ;6;(‘2’8’

ACCY~‘TCT~~~~ uutiAAti~~kt&~m~ ew

T~~~~~ATAT~T~~A~~~~TA~~~~~~

Fig. 1. Nucleotide sequence of CDC2a and CDC2b cDNAs and deduced aa sequences. Nucleotide No. 1 corresponds to the beginning of the longest cDNA. Poly(A) tracts in the longest CL)C2a and CDCZb cDNA clones were 28 and 20 nt long, respectively. In two shorter CDC2a cDNA clones, poly(A) addition followed nt 1276 and 1296, respectively. Last digits of numbers are aligned with corresponding nt. The numbers in parentheses on the right indicate the aa residues numbered in respect to the first residue. Transiation start and stop codons are shaded. The sequence data shown will appear in the EMBL, GenBank, and DDJB Nucleotide Sequence Databases under accession Nos. X57839 and X57840. Materials and Methods. An A. rhaliana

(Columbia ecotype) cDNA library constructed with lgtl0 vector (Huynh et al., 1986) was a gift of M. Learned (MIT., Cambridge, MA). Plaques from the library were transferred to BA85 nitrocellulose filters (Schleicher & Schuell), and were screened under hybridization conditions ofmoderate stringency (Matsui et al., 1989). The probes used for isolation of CDC2a and CDC2b cDNAs were the I.l-kb HindHI-KpnI fragment of pCdc2-5 (Durkacz et al., 1985) and the 0.63-kb Hind111 fragment of the CL>C2n cDNA clone, respectively. The A. thuliana DNA inserted in positive clones was subcloned in M13mp18 and/or M13mp19 (Yanisch-Perron et al., 1985) and sequenced for both strands by the chain-termination procedure (Sanger, 1981). The existence of CDC2a in A. thaliuna Landsberg ecotype was confirmed using polymerase chain reaction as follows. Two oligos were synthesized as primers with a Beckman System 1 DNA synthesizer. Their sequences, S’-CAPyCGTGAPyCTPyAAGCC and 5’-TCTGGGGCACGGTACCA, were designed to complement nt sequences encoding two separate parts of Skpombe p34’*” (HRDLKP for forward primer and WYRAPE for reverse primer). Ampii~~ation was done on an A. thafiana cDNA mixture with a DNA thermal cycler (Perkin EImerjCetus Corp.). After 45 cycles, the reaction products were treated with phenol, and the amplified DNA fragments were inserted at the HineII site on pGEM-3Z (Ph~a~ia) and sequenced. The cDNA mixture of A. thaliana was prepared as described in the legend to Fig. 2.

161

SC. pombe cdc2 gene probe. Hybridization signals (eight plaques) were detected at a frequency of 5 x 10 _ ‘. The size of A. thafiuna DNA inserted in these phages varied with the clone, but their restriction patterns overlapped (data not shown). Therefore, all clones were likely to be derived from the same species of mRNA. The nt sequence of the longest insert (1.43 kb; CDC2a) contained an ORF and a poly(A) tract (Fig. 1, CDC2a). Two shorter clones had the corre- sponding identical sequences except for polyadenylation sites following nt 1276 and 1296, respectively. Though the 3’-noncoding region was A + T-rich, there was no typical signal sequence (AATAAA) for poly(A) addition (Proudfoot, 1991). Northern-blot analysis with a probe of the CDCZa coding portion gave a hybridization signal at the 1.5-kb position (Fig. 2), indicating that the longest clone is nearly full-length and that the first ATG is likely to be the actual start codon. The deduced aa sequence with 294 residues (M, 34028) has similarity to all known ~34”““‘~ kinases as shown in Fig. 3: 63% of its aa are identical and 82% are similar (identical and conservative changes) to SC. ~ombe ~34~~~~ (IIindley and Phear, 1984); 6.5% identi- cal and 82% similar to human ~34~~” (Lee and Nurse, 1987); and there is a higher similarity (89% identical) to the putative ~34~~~~ from garden pea (Pisum sativum), a partial sequence of which was recently published (Feiler and

Jacobs, 1990). Furthermore, the ~34’~“~ hallmark ‘PSTAIR’ sequence motif (aa45-50) is completely con- served in this protein. According to Western-blot analysis with a protein sample that was prepared from induced Escherichia co& cells carrying the CDC2a cDNA expressible from the tat promoter (De Boer et al., 1983), a 34-kDa protein and several smaller proteins (possibly degradation products of the former) reacted with rabbit anti-p34cdc2 antibody (raised against an oligopeptide, EGVPSTAI- REISLLKE) (data not shown). It was thus confirmed that the CDC2a cDNA can actually code for a p34cdc2-like pro- tein. A sequence identical to nt 548-659 of CDC2a cDNA was also identified with another A. thaliuna strain

Fig. 2. Autoradiograms of Northern-blot hybridization with a probe of CDC2a or CDCZb. Methods. Plants were grown under standard condi- tions at 22°C where 18-h illumination and 6-h darkness alternate. A. ~~I~~a RNA was isolated from whole plants by a method combining two described procedures (Ausubel et al., 1987; Murray and Thompson, 1980). Briefly, tissues that were quickly frozen in liquid nitrogen were ground with a mortar and pestle to a fine powder, from which RNA was extracted by the hot phenol procedure followed by cetyltrimethyl- ammonium bromide precipitation and CsCl density gradient centrifu- gation. Poly(A) + RNA was separated from poly(A) - RNA through oligo(dT)-cellulose (Pharmacia) column chromatography, and then fractionated on a 1.2% agarose gel (4 pg RNA/lane) containing 1.8% formaldehyde with running buffer of 20 mM MOPS Good’s buffer pH 7.0/5 mM EDTAj8 mM Na 9 acetate that was previously treated with diethyl pyrocarbonate. The size markers of RNA (Bethesda Research Laboratories) were run in parallel. RNA was transferred to an Immobilon-N PVDF membrane filter (Millipore Corp.), and hybridiza- tion was performed at 42°C for 20 h in a solution containing 40% fo~amide~5 x SSCjS x Denhardt mix (Ausubel et al., 1987)~lOO pg sal- mon sperm DNA per ml/OS% SDS/a probe DNA (about 5 x IO* cpm per yg). The filter was washed at 25°C twice for 30 min in 2x SSC/O.l% SDS and then twice for 30 min in 0.2 x SSC/Ol.% SDS. The autoradiograms were generated by a Fujix BAIOO Bio-Image Analyzer (Fuji Photo Film).

so ma 120 140

Fig. 3. Amino acid sequence comparison ofvarious ~34~‘~ kinases. CDC2a and CDC2b (see Fig. 1)A. thulium; Sp, SC. pombe(Hindley and Phear, 1984); SC, &I. cerevisiue (Lorincz and Reed, 1984); Hs, Homo sapiens (Lee and Nurse, 1987); and Ps, Pisum salivum (Feiler and Jacobs, 1990). Last digits of numbers are aligned with corresponding aa. Residues identical to those ofA. thaliana ~34~~“” are shaded. Dashes are introduced for the best matching. Similarity of each ~34’““‘~ with A. f~a~ju~u p34 CDCzl’ is represented by % identical residues in parentheses.

162

(Landsberg ecotype) by amplification of cDNA through

polymerase chain reaction (see Fig. 1 legend). Therefore,

both ecotypes are likely to contain an identical CDC2a gene

or at least quite similar ones.

(b) Functional similarity between Arabidopsis thaliana

CDC2a and Schizosaccharomyces pombe cdc2 Since the cdc2 homologs from human, chicken, and fly

are able to complement the defects caused by SC. pombe cdc2 mutations (Lee and Nurse, 1987; Krek and Nigg,

1989; Lehner and O’Farrell, 1990),A. thaliana CDC2a also

might compensate for SC. pombe cdc2 mutations. To test

this possibility, two yeast temperature-sensitive cdc2 mutant

strains, one harboring pNH290 that contained the CDC2a cDNA inducible from the nmtl promoter and the other

harboring its vector pREP1 (Maundrell, 1990), were

incubated at 26’ C or 34’ C under inducing or noninducing

conditions. At the permissive temperature, both yeast

strains grew regardless of the culture conditions used,

though pNH290 carriers grew slightly more slowly than

pREP1 carriers (Fig. 4, 26°C). At the restrictive tempera-

ture, a significant increase in cell number was observed only

with pNH290 carriers cultured under the inducing condi-

tions; nevertheless, no considerable difference in the tur-

bidity was found between two strains under either set of

conditions (Fig. 4, 34°C). The cell morphology of the 7-h

samples in Fig. 4 is shown in Fig. 5. The elongated shapes

characteristic of cdc2 mutants were restored to normal by

introduction of pNH290 under the inducing conditions but

not under the noninducing conditions. These results indi-

cate that A. thaliana p34cDcz” and SC. pombe ~34’~‘~ are

similar not merely structurally but also functionally. It was

thus concluded that the CDC2a cDNA clone was derived

from a genuine A. thaliana CDC2 gene. However, after pro-

longed incubation at the restrictive temperature, the number

of pNH290 carriers under the inducing conditions reached

a plateau, and a similar growth inhibition was detected with

a cdc2 + strain harboring pNH290, but not harboring

pREP1, under the inducing conditions (Fig. 4, 34°C).

Therefore, complementation between the A. thaliana CDC2a and SC. pombe cdc2 genes seems to have occurred

incompletely and/or merely in a limited period of time, and

accumulation of A. thaliana ~34~~“” over SC. pombe p34”dS appears to be deleterious to yeast cells. Consistent

with this, we have not succeeded in constructing SC. pombe strains in which the CDC2a cDNA is transcribed from a

constitutive promoter such as the adh promoter (T.H.,

unpublished results).

The partial complementation, observed may imply that

A. thaliana p34 CDC2a is functional in SC. pombe only at the

G2-M transition for the following reasons. As seen in

Fig. 3, three residues (Thr14, Tyr”, and Thr16’), the phos-

phorylation and dephosphorylation of which modulate

NO. of Cdl8 (26 %)

No. of Cdl8 (24 %I

4 8

TutlMly (26 ‘c,

Turbidity (24%)

/

Fig. 4. Complementation of cdc2 function by A. thaliuna CDC2a cDNA

expressed in SC. pombe. Methods. A PvuI-SspI fragment that contained

the CDC2a coding region (nt 100-l 112 in Fig. 1) was inserted into the

BamHI site of pREP1 carrying the nmtl promoter inducible by thiamine

deprivation (Maundrell, 1990) after the sticky cleavage ends were tilled

in. The recombinant plasmid with a proper orientation (pNH290) and the

parental pREP1 were used to transform SC. pombe SP33 (leul cdc2-33’“)

and HM 123 (leul cdc2 + ) to Leu + on selective minimal agar (Ito et al.,

1983) at 26°C. The resulting four yeast strains were grown at 26°C to late

log phase in EMM2 medium (Maundrell, 1990) supplemented with 2 PM

thiamine, and then diluted into EMM2 without (inducing conditions) or

with thiamine (noninducing conditions). After incubation at 26°C for

14 h, the temperature was raised to 34°C (O-time), while control cultures

were kept at 26°C. At intervals, the turbidity of cultures was measured,

and the number of cells counted under a microscope. During these

cultivations, the cell density was always maintained at less than about

3 x 10’ cells/ml by dilution. The abscissa shows incubation time in h after

O-time, and the ordinate represents the logarithm of the number of cells

(left panels) or the turbidity (right panels) relative to those at O-time,

which were about 5 x lo6 cells/ml and about 0.1 of A,,,. Open and

blackened circles, SP33[pNH290] under inducing and noninducing con-

ditions, respectively; open and blackened triangles, SP33[pREPI] under

inducing and noninducing conditions, respectively; open squares,

HM123[pNH290] under inducing conditions; plus symbols,

HM123[pREPl] under inducing conditions.

p34=d=* activity at the G2-M transition (Lewin, 1990), are

completely conserved in this protein. On the other hand, the

p34cdc2-distinctive Se?“, the phosphorylation of which

peaks during Gl phase and drops markedly on entering S

phase (Krek and Nigg, 1991), is replaced by Asn2”. If the

Ser2” dephosphorylation function of ~34”~‘~ kinase is es-

sential in the Gl-S transition, A. thaliana ~34~~~~” must

naturally be lacking one of the SC. pombe ~34”~“~ dual

functions.

(c) Arabidopsis thaliana may contain a CDC2 gene family

To see whether cognate genes similar to CDC2a exist in

the A. thafiana genome, the cDNA library was again

163

Fig. 5. Morphology of cdc2 mutant cells carrying pNH290 or pREP1. Cells at 7-h incubation in Fig. 4 were fixed in 10% formalin and photographed. (a) SP33[pREPl] incubated at 26°C under inducing conditions; (b) SP33[pREPl] incubated at 34°C under inducing conditions; (c) SP33[pNH290] incubated at 34°C under inducing conditions; and (d) SP33[pNH290] incubated at 34°C under noninducing conditions. SP33[pNH290] at 26°C under either set of conditions showed similar shapes to those in (a)/(c), and the morphology of SP33[pREPl] at 34°C under noninducing conditions was analogous to that in (b)/(d).

screened with the CDC2a cDNA probe under mild-

stringency conditions. Plaques giving clear hybridization

signals were CDC2a cDNA clones, while those with weak

signals (four clones) that were detected at a frequency of

1 x 10 - ’ contained other kinds of cDNA. From restriction

pattern analysis (data not shown), all of the latter clones

seemed to be derived from the same mRNA species

(CDC2b). Their sequences (Fig. 1, CDC2b) contained one

ORF and 3’-noncoding A + T-rich regions, but no typical

poly(A) addition signal, as in the case of CDC2a. The

predicted aa sequence (170 aa deduced from the longest

0.73-kb clone) is similar to the corresponding regions of

both A. thaliana ~34~~“” (61% identical) and SC. pombe p34=dc* (60% identical), while there were 67% identical

residues within this limited region of A. thaliana ~34~~~~” and SC. pombe ~34”~~~. Thus, the A. thaliana CDC2b gene

product is more divergent from SC. pombe ~34’~‘~ than is

A. thaliana ~34~~~~“. However, two p34cdc2-characteristic

aa residues within the sequenced region, Thr16’ and Ser2”

(Lewin, 1990), the latter of which is not conserved in

P34 cDc2u, are found in the CDC2b gene product. As the

deduced ORF continues to the 5’ end of the longest cDNA

and a translation start codon has not been identified, the

sequence does not seem to be full-length. Indeed, Northern-

blot analysis showed the CDC2b mRNA to be about 1.4 kb

(Fig. 2). Since this size is close to that of the CDC2a mRNA, CDC2b also appears to code for a p34cd’2-like

protein. Attempts have been unsuccessful at obtaining

longer cDNA clones, and all of the isolated cDNAs ended

within a narrow region, suggesting that the RNA may have

a secondary structure that prevents extension beyond this

region.

To find out whether the CDC2 genes are dispersed over

the A. thaliana genome, genomic Southern-blot analysis

(BamHI and EcoRI digests) was done with CDC2a and

CDC2b cDNA probes under high- and low-stringency con-

ditions (Fig. 6). Under the high-stringency conditions, each

probe made one or two specific DNA fragments visible.

Under the low-stringency conditions, on the other hand,

several additional weak bands were detected. One such

band probed with CDC2b cDNA corresponded to the clear

band probed with CDC2a cDNA under the high-stringency

conditions, and vice versa. Therefore, the A. thaliana ge- nome seems to contain one copy each of CDC2a and

164

Fig. 6. Autoradiograms of genomic Southern-blot hybridization. Methods. A. thaliana DNA was isolated from whole plants using cetyltrimethylam-

monium bromide as described (Murray and Thompson, 1980), and cleaved with BarnHI or EcoRI (as shown above the lanes). DNA fragments in the

digests were fractionated on 0.7% agarose gels and then transferred to membrane filters as described in the legend to Fig. 2. Both high- and low-stringency

hybridization conditions were used (as shown above the lanes). The former conditions were the same as in Northern-blot hybridization except for a single

wash at 60°C instead of two washes at 25°C in 0.2 x SSCjO.1 y0 SDS. In the latter conditions, the formamide concentration in the hybridization solution

was lowered to lo%, and all washes were done at 25°C. ‘CDC2a’ and ‘CDC2Y indicate the probe DNA, and ‘High’ and ‘Low’ represent the high- and

low-stringency hybridization conditions, respectively.

CDC2b and also several other cognate genes. These genes

may constitute a CDC2 gene family. The respective CDC2

genes may have separate functions because the ~34’~“’

-specific Ser2” is conserved in the putative ~34~~~~’ but

not in p34cDc2” as described in section b. This situation is

probably comparable to the case of Drosophila where only

one of the two cdc2 homologs can replace SC. pombe cdc2

(Lehner and O’Farrell, 1990).

(d) Conclusions

We demonstrated that the A. thafiana genome contains

two different genes which are similar to the SC. pombe cdc2

gene. Besides, the presence of more cdc2 homologs was

suggested. They may constitute a CDC2 gene family in

A. thaliuna. One of the member genes, CDC2a, resembled

SC. pombe cdc2 functionally as well as structurally, sup-

porting the view that there is the p34”d’2-centered universal

system for cell-cycle control in every eukaryote. Clarifying

whether the function of each member gene is different from

or overlaps with another would provide information on the

control system of the mitotic cell cycle in A. thaliuna.

ACKNOWLEDGEMENTS

We are grateful to Drs. N. Goto and K. Okada for their

generous gifts of A. thaliuna seeds, M. Learned for an

A. thaliana cDNA library, P. Nurse for pCdc2-5, T. Takeya

165

for anti-p3pdc2 antibody, and T. Toda for pREP1 and SC. pombe strains. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

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