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The Plant Cell, Vol. 7, 1143-1155, August 1995 O 1995 American Society of Plant Physiologists Distinct Classes of Mitotic Cyclins Are Differentially Expressed in the Soybean Shoot Apex during the Cell Cycle Hiroshi Kouchi,ail Masami Sekine,b and Shingo Hataa92 a Department of Applied Physiology, National lnstitute of Agrobiological Resources, Tsukuba, lbaraki, 305 Japan Department of Biotechnology, Osaka University, Suita, Osaka, 565 Japan Protein phosphorylation by the complexes of cyclin and cyclin-dependent kinase plays a key role in cell cycle progression in all eukaryotes. The amplification by polymerase chain reaction of a cyclin box from developing root nodules and root apices of soybean showed the expression of a number of different molecular species of mitotic cyclins in plant meristems, and they were classified into five distinct groups based on their sequence similarities. The complete soybean cyclin cDNAs, cyclGm to cycSGm, corresponding to each group were isolated, and their predicted amino acid sequences showed clear similarities to mitotic cyclins identified from various organisms. These genes are expressed predominantly in such meristematic tissues as root and shoot apices and young developing nodules. Double-target in situ hybridization involving histone H4 as an S-phase marker allowed us to estimate the phases during which these cyclin genes are abundantly expressed. The results indicated that cyc5Gm is expressed in GP-to-Mphases and cyc3Gm is expressed from late S-toG2 phases. These expression patterns, together with the sequence criteria, strongly suggest that cyc3Gm and cyc5Gm encode the plant cognates for A- and B-type cyclins, respectively. In addition, the expression of cyclGm was restricted during a short period in S phase, suggesting that it belongs to a nove1 class of plant cyclins. Sequence comparison of 18 plant mitotic cyclins cloned thus far showed that they can be divided into four distinct structural groups with different functions in cell cycle progression. INTRODUCTION The cell division cycle in eukaryotes is controlled by common mechanisms in which heterodimeric protein kinases play the central role. These protein kinases consist of the catalytic subunit, cyclin-dependent kinases (cdks), and their regulatory subunit, cyclins. The association of the cdks with specific cyclins, together with phosphorylation and dephosphorylation of cdks, is required for most eukaryotic cell cycle transitions (for reviews, see Norbury and Nurse, 1992; King et al., 1994; Nurse, 1994). Recent advances in the study of the eukaryotic cell cycle have shown the presence of a number of structurally homologous cdks and cyclins in higher eukaryotes. Among them, the role of the association between p34CdC2, the 34-kD protein kinase encoded by the fission yeast cdc2 homologs, and mitotic cyclins is the best understood. Cyclins were first identified in marine invertebrates (Evans et al., 1983; Swenson et al., 1986); the p34cdc2-cyclincomplexes must function for cells to undergo mitosis, that is, to enter into M phase. The M-to-G,phase transition is accompanied by the dissociation of these complexes and the breakdown of cyclins (for review, see Hunt, 1991; Surana et al., 1993; King et al., 1994). To whom correspondence should be addressed., * Current address: Faculty of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606 Japan. The animal mitotic cyclins have been subdivided into A- and B-type cyclins, according to differences in their sequences and expression patterns during the cell cycle. Although A- and B-type cyclins share many structural and functional similarities, they do not appear to be redundant in cell cycle regulation in many vertebrates. 60th have been implicated in the G2-toM-phase transition, activation of the p34cdc2protein kinase, and induction of the entry into M phase (Minshull et al., 1989). However, A-type cyclins appear to piay a role in DNA replication during S phase, in addition to their role in progression through GP phase (Girard et al., 1991; Pagano et al., 1992; Zindy et al., 1992). It has also been suggested that the substrate specificity of cyclin-cdk complexes is differentially modulated by A- and B-type cyclins (Peeper et al., 1993). In addition, it has been suggested that even at the Gz-toM-phase transition, A- and B-type cyclins carry out different functions, because in Drosophila, the B-type cyclin is unable to compensate functionally for the loss of the A-type cyclin (Lehner and OFarrell, 1990). The S-phasefunction of the A-type cyclin probably involves the formation of a complex with p33cdk2,which is structurally related to but functionally distinct from p34cdc2,and has been implicated in G, and S phases (Pines and Hunter, 1990; Pagano et al., 1992). In Saccharomyces cerevisiae, no cyclin A structural counterpart has been identified. However, CLB5 and CLB6 have been suggested 1144 The Plant Cell to be functionally equivalent with A-type cyclins, because they operate in the timing and execution of S phase (Kühne and Linder, 1993; Schwob and Nasmyth, 1993). The other subfamilies of cyclins have been identified as G1 cyclins. They are required for completion of G1 phase and commitment to the entry into S phase. CLN1, CLN2, and CLN3 from S. cerevisiae were the first G1 cyclins to be identified (Nash et al., 1988; for review, see Reed, 1991). Thus far, mammalian C-, D-, and E-type cyclins have been identified as G1 cyclins (Lew et al., 1991; for review, see Sherr, 1993, 1994). In S. cerevisiae, CLNs and mitotic cyclins share p34CDC28 as the only catalytic subunit, whereas mammalian D- and E-type cyclins seem to form complexes with cdks other than p34cdc2 (Pines, 1993; Sherr, 1993,1994). With the exception of the human E-type cyclin (Lew et al., 1991), these G1 cyclins share no or very limited structural similarity with mitotic cyclins. Higher plants grow by continuously differentiating new organs postembryonically from meristem cells, according to interna1developmental programs or as a result of environmental stimuli. New organs are normally initiated by the division of specific groups of cells even in mature plants, and only a few phytohormones are involved in these processes. Therefore, higher plants and animals must differ significantly in the control of the cell division cycle (Jacobs, 1992). We first identified plant cyclins from soybean and carrot (Hata et al., 1991). Subsequently, a number of cyclins have been described from a wide variety of plants, that is, Arabidopsis (Hemerly et al., 1992; Ferreira et al., 1994), alfalfa (Hirt et al., 1992), Antirrhinum (Fobert et al., 1994), maize (Renaudin et al., 1994), and tobacco (WSetiady, M. Sekine, N. Hariguchi, T. Yamamoto, K. Yoshida, H. Kouchi, and A. Shinmyo, unpublished data). These cyclins all share conserved “cyclin box” structures, and some of them are able to induce maturation of Xenopus oocytes (Hata et al., 1991; Renaudin et al., 1994) or complement a CLN mutant of S. cerevisiae (Y.Y. Setiady, M. Sekine, N. Hariguchi, T. Yamamoto, K. Yoshida, H. Kouchi, and A. Shinmyo, unpublished data). In addition, G1 cyclin candidates related to cyclin D have recently been isolated from Arabidopsis, and they complement S.cerevisiae CLN mutants (Soni et al., 1995). A number of functional and/or structural homologs of cdks have also been isolated from higher plants (Colasanti et al., 1991; Feiler and Jacobs, 1991; Ferreiraet al., 1991; Hashimotoet al., 1991; Hata, 1991; Hirayama et al., 1991; Hirt et al., 1991, 1993; Mia0 et al., 1993). These results show that a common mechanism of eukaryotic cell cycle regulation is probably conserved in higher plant cells. However, the available data on regulatory mechanisms of the cell division cycle in higher plants are still very limited. In plants, the cell division cycle can be arrested in either the G1 or the G2 phase; cells arrested at G1or G2 have the capacity to reenter the cell division cycle (van’t Hof, 1985; Bergounioux et al., 1992). Thus, the availability of mitotic cyclins as well as of G1 cyclins may be critical for regulating differentiation and/or dedifferentiation processes in plants. Further characterization of plant cyclins will provide more clues to the molecular mechanisms that control developmental processes in higher plants. In this study, we show that soybean plants express a number of different cyclins in the meristematic tissues. Among the five complete cDNAs isolated, three have A-like structures and the other two have B-like structures. Analyses of their expression in shoot meristems by in situ hybridization demonstrated that the transcription of these distinct classes of cyclin genes is regulated differentially during the cell cycle in plant meristems. RESULTS Cloning of Cyclin cDNAs Degenerated oligonucleotide primers were designed for a highly conserved region of published amino acid sequences of mitotic cyclins (Hata et al., 1991). Polymerase chain reaction (PCR) using these primers with the cDNAs from young developing nodules or root tips resulted in amplification of cDNA fragments with the expected length (185 bp). These fragments were subcloned, and 30 clones were sequenced. As a consequence, 29 clones showed the conserved amino acid sequences typical of mitotic cyclins (data not shown). These clones were tentatively divided into five groups (designated groups I toV) according to structural similarities. Groups I, II, and III contain four, three, and two different molecular species, respectively, whereas groups IV and V each consist of only one molecular species. The group V species has a nucleotide sequence identicalto s73-6, which previously was identified from soybean nodules (Hata et al., 1991). Therefore, we attempted to isolate full-length clones for groups I to IV by screening the nodule cDNA library using PCR products of each group as probes. Cloning of cDNAs of groups I and II from the nodule cDNA library was not successful, probably due to the Iow leve1 of expression of these cyclins in intact tissues. However, they could be isolated from a cDNA library of suspension-cultured cells. Finally, we obtained four soybean cyclin cDNA clones (cyclGm, cycPGM, cycSGM, and cyc4Gm) corresponding to groups I, II, III, and IV, respectively. Their deduced amino acid sequences are shown in Figure 1 with Cyc5Gm, which formerly was named S13-6 (Hata et al., 1991). Highly conserved amino acids in the cyclin box are well conserved in all sequences; moreover, all sequences contain a conserved destruction box in the N-terminal region that is responsible for ubiquitin-mediated proteolysis. conserved amino acids for A- and 6-type cyclins are shown above the soybean cyclin sequences in Figure 1 (Hata et al., 1991). These conserved amino acids appear to be distributed almost randomly among the five soybean cyclins, and it was not easy to assign them to either of the phylogenetic types. However, it was possible to classify them into two groups based on the homology over the entire amino acid sequences; one includes CyclGm, Cyc2Gm, and CycBGm, and the other includes Cyc4Gm and Cyc5Gm. The amino acid identities are 45 to 50% between CyclGm, Cyc2Gm, and Cyc3Gm, and 55% 1145 Differential Expression of Plant Cyclins * * A-con. B-con. CyclGm CycZGm Cyc3b cyc4Gm cyc5Gm A-con. R-cnn. CyclGm CycZGm Cyc3h Cy~4Gm Cyc5Gm M D K V N R V C A K D E E R P L R I T R R R A L R G I T P Y S R P S L K N E S K MSTQNRRSSFSSSlTSSLAKRHASASTTSSLAAAPTNMAKKRPPLSNLTNTVAHRNSSSSVPCA MASRLEQQQQ---PTNVGGENKQK-NMGGEGRNRRVLQDIGNLVGKQGHGNGI~S MASRIVQQQQARGEAVVGKQQKKNGVADGRNRKALGDIGNWRGVVDAKPN- EKLQCRKNPNA----------------------------------------------------------------------------------FBAKGVCTKKNTKLAASSVSTDVSSSHD--DVRAKLAEELSTIKMYESNDTLREGVTADTSLSMQNSVKSDELRNSPNKDIDIICEKLGASDS--AKFAKTKKDWPACSGNKRPKPASAVIFPKANSFPVKNEAPPTPPPPVATVTVPAPWDVSPSKSD~SVSTDESMSSCDSFKSPDI KPYTRNFRQLLANAaAAT--EKNKK-SSTEVNNGAVVATDGVGVGNFPARKVGAAKKPKEEPEVIVIISDDESDEKPAVKGKKAREKSAMKN RPITRSFGAQLLANAQAAAAADNSKRQACANVAGPPAVANEGVAVAK~PKPVSK~IVKPKPSEK~IDASPDKKEVLKDKKKEGD~PKK * A-con. B-con. CYCl& CYC2& Cyc3Gm Cyc4Gm Cyc5h I * ** $ MR IL DWLVEV EEYKL ETL L MR IL DW Q F LLQEI ** * *$* Y DRFLS I DR V R KLQLVG A V LQLVGYT CYC~G~ * * * **** *<* * A KYEEIYPP V EF A KYEEMY P DF * t ** * TDD Y Q LRME D YT I ME L L VTPIMRGILVDWLVEVAEEYKLLSDTLHLSVSYIDRFLSVNP~KSRLPLLGVSSMLIAAKYEETDPPSVDEFCSITDMYDKAEWKMEADIL I N P S M R G I L V D W L V E V S E E Y K L V P D T L Y L ~ L I D R n S T L INSSMRILIDWLVEVAEEYRLVPDTLYL~NYIDRYLSGNVMNRPRLPLLGVAS~IASKYEEICAPaVEEFCYITDNIYFKEEVLQMESAVL INAKMRSILVDWLIEVH_RKFELMPETLYLTLNIVDRFLSVKAVPRRELaLVGISSMLIASKYEEIWAPEVNDFCISDNGWSEQVLMMEKQIL INERMRILVDWLIDV~ELSLETLYLTINIIDRFWLVKTVPRRELQLVGISAMLWLSKYEEIWPPEVNDFCLSDR~~IL~~IL ** ** * A-con. B-con. CYclGm CYCZGII ** * Y I Y R E K K YM Q D D D P SEY I Y Y E Y E ............................................ NKPSPTNNTLSSPQL-DGSWSDIHEYLREMEMPKKRRPMVNYIEKFQKI -_--------_---_----_____________________---LTIVDIDSELKDPQL-WSFYAPDI--YSNIRVTELQRKPLTNYMDKLQKD EWDNSDVAAVDSIERKTFSHLNISDSTEGNICSREILVELEKGDKFVNYD"YADWL-CATFACDI--YKHLRASEAKKRPSTDFMEKIPKE AKAF----- -- ---------- ----SSVLSARSKAACGL---PRDFKNIDATDMDNELAAAEYIDDI- -YKFYKETEEDGCV-HDYblG-SQPD KSQHTL-- - -- - -- - - - - - - -- - - -TSVLTARSKAACGITNKPKEQII DIDASDVDNELAAVEYIDDI- -YKFYKLVENESRP-HDYIG-SQPE >>**>** *** * A-con. 8-con. CyclGm CycZGm Cyc3Gm Cyc4Gn Cyc5Gm $ R ALGVI N R ALGZI N METRAAAKRKANAAIWWEKQHPKRQRWLGELPNLQNL I VSK IQNPRK ** *$ ** * *<< * * K L FD FT F K EL L Y PS A LA W TY L GRPL LHFLRR SK HT AKYLMEL PSIAA L L W Y Y E KSLKFEMGNPTVSTFLRRYAASDVQ~NSQIEHLGSYIGELSLLD~CL-RFLPSIVAASVIFLAKFIIWPEVHP~SSLCECSGYKPAEL N L V H F Q L S V F T I K T F L R R F I a A A Q S S Y K A P Y V E L E F L A N Y NFLKF~APNKCFLRRFVRGVDEVPSLQLECLTNYIAELS~EYSM-LGYAPSLVMSAIFW\KFILFPSKKPWNSTL~Y CYc4Gm CYc5Gm RKLEWn~PTPYHFLVRSTPSDK----EME~FFW\ELGLMHYPNILYRPSLIAASAVFAARCRSPF-~GYSEE~ A-con. B-con. CYC 1Gm CYc24 CYC~G~ Cyc4Gn C~c5Gm EKY Y V VKY S KECVLI LHDLYLSRKAAS- FKAVREKYKHQKFKCVANLPTPPYVPSCYFEDB KTVVLALQDLQLNTKGCF-LNAVREKYKQQKFNCVANLSPKSVQSLFQNQV CVCVKDLHRLCCNSPNSN-LPAIREKYSQHKYKYVAKKYCPPSIPPEFFQN N K L E ~ L ~ ~ L V F L V R F I K A S V P D Q - - - - - E L D N W * L A ** QA RDCAKIbL4NLHAAAAPGSKLRAVYKKFSNSDLSAVALLSPAKDLSALS MDCARLLVGFYSTLENG-KLRVVYRKYSDWKGAVAVLPPA~LLPEGSASQHS 348 469 484 440 454 Figure 1. Deduced Amino Acid Sequences for Soybean Cyclins. Conserved amino acids in A- and 8-type cyclins (A-con. and B-con., respectively) are shown above the soybean cyclins. The destruction box is underlined with a thick bar, and the cyclin box is bordered by the symbols >> and <<. Residues identical in all soybean cyclin sequences are indicated by asterisks. The region corresponding to the PCR products is bordered by the symbols > and <. The sequence motifs referred to in the text are underlined. Gaps (indicated by dashes) were introduced to optimize the alignment. The nucleotide sequences of qclGm, W C ~ G ~ , cyc3Gm, and cyc4Gm have been submitted to the GenBank, DDBJ, and EMBL nucleotide sequence data bases with the accession numbers D50868, D50869, D50870, and 050871, respectively. cyc5Gp (X62820) was formerly named s73-6 (Hata et al., 1991). 1146 The Plant Cell between Cyc4Gm and CycSGm. However, the identities between these two groups are only 23 to 29%. The amino acids conserved commonly in A- and B-type cyclins are highly conserved in all soybean cyclin sequences (81 to 94%). The amino acids conserved distinctively in A-type cyclins are conserved 63 to 73% in CydGm, Cyc2Gm, and CycSGm, whereas they are conserved 51 and 37% for Cyc4Gm and CycSGm, respectively. On the other hand, the amino acid consensus only for B-type cyclins is less well conserved in all five sequences, but they accounted for 23,41, and 23% for CydGm, Cyc2Gm, and CycSGm, respectively, and 43 and 45% for Cyc4Gm and CycSGm. In addition, the motif EVXEEY(K/R)L in the cyclin box typical of A-type cyclins is perfectly conserved in CydGm to CycSGm. Although the motif typical of B-type cyclins, FLRRXSK, is conserved more loosely in all five sequences, the motif HX(K/R)F, which is highly conserved in yeast B-type cyclins (Kiihne and Linder, 1993), is present in both Cyc4Gm and CycSGm. Thus, CydGm, Cyc2Gm, and CycSGm resemble A- rather than B-type cyclins, whereas Cyc4Gm and CycSGm more closely resemble B-type cyclins. However, these sequence criteria were not enough to assign them definitely to either of the phylogenetic types (Hata et at., 1991; Ferreira et al., 1994). The nucleotide sequence homologies among these five cyclins are 40 to 60%, and they do not cross-hybridize each other under the standard high-stringency conditions. Therefore, we used the entire cDNA inserts for DNA and RNA gel blot analyses. Figure 2 shows gel blot hybridization of soybean genomic DNA with each cyclin cDNA insert. The results indicated totally discrete patterns of hybridization, and, therefore, these five cyclin genes are not tandemly arranged. cydGm cyc2Gm cycSGm cyc4Gm cycSGm E H E H E H E H E H 23.1- 9.4- •* 6.64.3- 1 2 3 4 5 cydGm (1.5) cyc2Gm (2.8) cycSGm (18) cyc4Gm (1.7) * cycSGm (1.7) ccfc2 (1.4) Figure 3. RNA Gel Blot Analysis of Total RNA Isolated from Various Tissues of Soybean Seedlings with Cyclin cDNAs. Total RNAs (10 ng) were electrophoresed on a 1% agarose gel, transferred to nitrocellulose membranes, and probed with the cDNA inserts indicated. The numerals within parentheses indicate the length of the transcripts in kilobases. Lane 1, shoot apex; lane 2, root tips; lane 3, nodulated root segments at 4 days after B. japonicum inoculation; lane 4, stems; and lane 5, nodules at 9 days after inoculation. 2.3- Expression of Cyclin Genes in Various Tissues 1.050.770.56- Figure 2. DNA Gel Blot Analysis of Genomic DNA Isolated from Soybean Hypocotyls. Ten micrograms of DNA was digested to completion with EcoRI (E) or Hindlll (H), electrophoresed on a 0.8% agarose gel, transferred to nylon membranes, and probed with the cDNA inserts indicated. Numbers at left indicate the lengths in kilobases. Figure 3 shows the results of gel blot hybridization of total RNAs from various organs of soybean seedlings. All five cyclin transcripts were predominantly expressed in the meristematic organs, such as shoot apices, root tips, and young developing nodules, but at very low levels in stems (control nonmeristematic organs), indicating that the expression of these genes is closely related to cell division. These patterns of cyclin expression contrasted with that of cdc2, which was highly expressed in stems as well as in meristematic organs. The cdc2 probe that we used has a sequence 99% identical with cdc2-S5 (Miao et al., 1993) containing a PSTAIRE domain. Recently, Differential Expression of Plant Cyclins a nove1 member of the cdc2 gene family has been described that does not contain a perfectly conserved PSTAIRE domain and shows the cell cycle-dependent expression pattern (Forbert et al., 1994). The unusually large sizeof cyc2Gm mRNA is due to the presence of a M O 0 nucleotide noncoding sequence at the 5' end. The transcript levels of cyc5Gm were highest in all organs, whereas cyc7Gm and cyc2Gm transcripts were at very low levels. The transcripts of cyclGm and cyc5Gm appeared to be most abundant in shoot apices, whereas those of cyc3Gm were more abundant in root tips and nodules. However, these differences are not enough to demonstrate the differentialexpressionof cyclins betweendifferent meristematic tissues, and we conclude that these five cyclin genes are all expressed in the meristematic organs of soybean seedlings. Cell Cycle-Specific Expression of Cyclin Genes In situ hybridization of soybean shoot apices with histone H4, cyc7Gn-1, cyc3Gm, and cyc5Gm is shown in Figures 4A to 4D, respectively. Experiments with cyc2Gm and cyc4Gm were unsuccessful because of the extremely low hybridizationsignals. The scattered signal appearance in isolated cells in the meristematic region is a characteristic feature of cell cycle-specific genes (Fobert et al., 1994). The signals appeared randomly in isolated cells in the apical meristems, vascular cambium, and developing leaves. The frequency of labeled cells and the signal intensity were highest for histone H4, intermediate for cyc5Gm and cyc3Gm, and very low for cyc7Gm. In particular, the hybridization signals for cyc7Gm appeared in only a small fraction of the cells in the shoot apex, indicating expression during a short period of the cell cycle. The differences in the frequencies and relativesignal intensities betweenthese genes were almost consistent with the results of the RNA gel blot hybridization analysis (Figure 3). Differential Expression of Cyclin Genes during the Cell Cycle To examine the interrelationships among expression of these genes, we performed double-target in situ hybridization using a combination of any two probes involving histone H4, according to the method developed by Fobert et al. (1994). Because transcripts of histone genes accumulate preferentially and abundantly in S phase (Nakayama and Iwabuchi, 1993),they provide a good control to mark S-phase cells. The microtome sections of shoot apices were hybridized with a mixture of digoxigenin- and fluorescein-labeled probes, and the signals were detected sequentially using anti-digoxigenin and antifluorescein antibodies conjugated with alkaline phosphatase in combination with different color substrates (see Methods). A typical result for a combination of cyc5Gm and histone H4 is shown in Figures 5A and 58. The sections were hybridized with a mixture of digoxigenin-labeled cyc5Gm probe and 1147 fluorescein-labeled histone H4 probe. Hybridization signals for cyc5Gm were first detected using anti-digoxigenin-alkaline phosphatase conjugate with the Fast Red substrate (Figure 5A). After heat inactivationof alkaline phosphatase conjugated to anti-digoxigenin,signals for histone H4 were visualized using anti-fluorescein-alkaline phosphatase conjugate with nitro blue tetrazolium salt/5-bromo-4chloro-3-indolyl phosphate toluidinium salt (Figure 5B). Signals for the cyc5Gm probe appeared in cells completely different from those containing signals for the histone H4 probe. We examined more than 250 cells from a number of independent sections showing the cyc5Gm signals, and we found that only 2% of them expressed histone H4. Similar experiments were performed for combinations of cyc3Gm and histone H4 (Figures 6A and 6B) and combinations of cyc3Gm and cyc5Gm (Figures 6C and 6D). The expression of cyc3Gm overlapped that of histone H4 in part (9% of 250 cells with cyc3Gm signals showed histone H4 expression), whereas it overlapped with that of cyc5Gm in a much higher proportion(41% of 255 cells). The results of experiments that used cyc7Gm were contrasted with results of those that used cyc3Gm. The cells with cyc7Gm signals were completely included in those with histone H4 signals (Figures 7A and 76), whereas essentially no cells expressed both cyc7Gm and cyc5Gm (Figures 7C and 7D). Figures 8A to 8F show the results of in situ hybridization with %-labeled probes for histone H4, cyc5Gm, and cyc3Gm. The sections were counterstained with toluidine blue, and dividing cells at metaphase and early telophase could be distinguished. However, it was difficult to distinguish the cells at prophase from those at interphase due to the limitation of toluidine blue staining. Signals for histone H4 were very strong, but dividing cells at metaphase and early telophase contained no hybridization signal (Figure 8A). In contrast, the signals for cyc5Gm were clearly detectable in cells at metaphaseand interphase (and prophase). During early telophase, the cells showed weak signals that were significantlyabove the background leve1 (Figures 8B and 8C). For cyc3Gm, the hybridization signals were barely detectable in dividing cells at metaphase and early telophase and appeared only at interphase (Figures 8D to 8F). DlSCUSSlON In this study, we describe the characterization of five distinct mitotic cyclins from soybean plants, cyc7Gm to cyc5Gm. The expression patterns of three of these, cyclGm, cyc3Gm, and cyc5Gm, were analyzed in further detail by double-target in situ hybridization involving histone H4 as an S-phase marker. As described first by Fobert et al. (1994), the transcripts of these cell cycle-related genes appeared in isolated cells dispersed in the meristematic tissues (Figures 4A to 4D), indicating that they are expressed during limited periods in the cell cycle. Similar dispersed patterns of expression of cyc2Ms and histone H4 have been described in nodule meristems of alfalfa (Yang M: • "*fc%\*. ,.\ • .» '. ™ i!fei$L. -f/c^.1 - - rJiw ••"-' •; -v "-V /,.pi •,:-.'• r;.-••-'• v. -^i -^ ' ^ V J "" ^'A'*.;*^/-iC* ^". . v - ' - 4 i Vjaag^ .,> •';>> ^» l *• ' . ""5. Figure 4. Localization of the Transcripts of Cell Cycle-Related Genes in Soybean Shoot Apices. Hybridization signals are visible as purple or dark blue color development. The sense strand control hybridization gave no signal (data not shown). (A) Histone H4. (B) cycSGm. (C) cyc3Gm. (D) cydGm. Bar = 200 urn. Differential Expression of Plant Cyclins 1149 Figure 5. Double-Labeling of a Soybean Axillary Bud for cycSGm and Histone H4. The paraffin-embedded sections were hybridized with the mixtures of digoxigenin- and fluorescein-labeled antisense RNAs. (A) Detection of hybridization signals with the digoxigenin-labeled cycSGm probe (red). (B) The same section after detection of the fluorescein-labeled histone H4 probe (black). Bar = 25 urn. et al., 1994). The frequencies of cells labeled by these probes should reflect the relative length of the phases in the cell cycle during which they are abundantly expressed. The frequencies of cells expressing cycSGm and cycSGm were considerably less than the frequencies of those expressing histone H4, and the expression of cydGm was found in an extremely small number of cells in the shoot apex. Although the signal intensities of in situ hybridization are not always parallel with the abundance of the mRNAs in vivo, the results show that the expression of cydGm is restricted to a very short period in the cell cycle. Double-target in situ hybridization showed more precisely the relative timing of the expression of these genes. cycSGm was expressed in cells different from those accumulating histone H4 mRNAs, indicating that the expression of cycSGm is temporally separated from S phase (Figures 5A and 5B). Moreover, transcripts of cycSGm were detected in dividing cells at metaphase and early telophase as well as in cells at interphase (Figures 8B and 8C). Therefore, assuming that these cell cycle genes are expressed once per cell cycle, it is most likely that cycSGm is expressed during G2 and early M phases. Similar results were obtained for two Antirrhinum cyclins, cydAm and cyc2Am, which have high homology with cycSGm (Fobert et al., 1994). Based on the G2- and M-phase-specific expression of cycSGm, we can deduce the phases during which cydGm and cycSGm are expressed. Transcripts of cyc3Gm were detected in a small fraction of the cells that expressed histone H4 and in a much greater proportion of the cells that expressed cycSGm (Figures 6A to 6D). In addition, they were barely detected at metaphase and telophase (Figures 8D to 8F). A simple explanation for these results is that cycSGm is expressed predominantly during late S and G2 phases. In contrast with the expression pattern of cycSGm, cydGm expression appeared to be restricted to S phase and did not overlap that of cycSGm (Figures 7A to 7D). We conclude that, taken together with the large difference in the frequencies of cells labeled with cydGm and histone H4, the transcripts of cydGm become abundant during a very short period in S phase. Several authors have described the sequences of A-like and B-like cyclins from higher plants (Hataetal., 1991; Ferreiraet al., 1994; Renaudin et al., 1994). In this study, we describe three A-like and two B-like cyclin sequences from soybean plants. Although these sequences show significant similarities with both A- and B-type cyclins, their similarities with amino acids conserved distinctly in A- and B-type cyclins are not as high, making it difficult to assign them to either the A- or B-type cyclin group based on sequence motifs alone. It has been demonstrated that A-type cyclins are synthesized and destroyed earlier in the cell cycle than are B-type cyclins (Minshull et al., 1990; Pines and Hunter, 1990). Double-target in situ hybridization studies with cycSGm, cycSGm, and histone H4 clearly indicate that cycSGm is expressed at a much earlier phase of the cell cycle than is cycSGm. Therefore, the results presented in this study strongly suggest that cycSGm and cyc5Gm are A- and B-type cyclins, respectively, based on their differential expression patterns during the cell cycle, in addition to the sequence criteria. Furthermore, we have recently isolated three mitotic cyclin cDNAs from tobacco, Ntcyc25, Ntcyc27, and Ntcyc29, that are homologous with cycSGm, cyc2Gm, and cycSGm, respectively (YY. Setiady, M. Sekine, N. Hariguchi, T. Yamamoto, K. Yoshida, H. Kouchi, and A. Shinmyo, unpublished data). Their sequence identities across ~170 amino acids of the cyclin box are 88% 1150 The Plant Cell Figure 6. Double-Labeling of Shoot Apices with cyc3Gm and Histone H4, and with cyc3Gm and cycSGm. The arrows indicate the positions of cells with hybridization signals from both probes. (A) and (B) Detection of hybridization signals with the digoxigenin-labeled cycSGm probe (red) followed by that of the fluorescein-labeled histone H4 probe (black in [B]). (C) and (D) Detection of hybridization signals with the digoxigenin-labeled cyc3Gm probe (red) followed by that of the fluorescein-labeled cycSGm probe (black in [D]). Bar = 25 urn. for Ntcyc25 and Cyc3Gm, 86% for Ntcyc27 and Cyc2Gm, and 77% for Ntcyc29 and CycSGm. Their expression patterns in the highly synchronized culture of tobacco suspension cells clearly indicate that A/fcyc25 and A/fcyc27 are expressed predominantly during S-to-G2 phases, whereas Ntcyc29 is expressed during G2-to-M phases. These findings again suggest that the classes of cyclins represented by cycSGm and cycSGm are, respectively, the A- and B-type cyclin cognates from higher plants. The expression pattern of cydGm is quite unlike that of cycSGm and cycSGm. The sequence of cyclGm is more closely related to A- than to B-type cyclins, but expression is restricted to a short period of S phase and does not appear to be associated with G2 phase. Therefore, cydGm might represent a novel class of cyclins with a function in DNA replication during S phase. Since the first evidence of plant cyclins (Hata et al., 1991), 18 plant cyclins have been described, including the four soybean cyclins newly identified in this study. Renaudin et al. (1994) and Ferreira et al. (1994) have shown that plant mitotic cyclins can be divided into at least three groups, one consisting of A-like cyclins and the other two of B-like cyclins. More sequence data are now available, particularly for A-like cyclins, and we constructed a phylogenetic tree of these cyclins from a multiple sequence alignment of the cyclin box (Figure 9). This revealed that the plant cyclins identified so far can be divided into two distinct groups. One presumably includes A-type and the other B-type cyclins. Each group appears to be subdivided into two classes, which we tentatively designated a1, a2, (31, and (52, according to the terminology proposed by Soni et al. (1995). (31 and P2 cyclins both have a well-conserved HX(K/R)FL motif within the cyclin box, but a more upstream conserved Differential Expression of Plant Cyclins (S/T)(S/T)VL(S/T)ARSKAACG motif (Renaudin et al., 1994) was found only in P1 cyclins. a1 and a2 cyclins have a conserved EVXEEY(K/R)L motif or closely related sequences within the cyclin box, which is typical for A-type cyclins. They also contain a few short conserved sequences upstream of the cyclin box. There are only two a1 cyclins, and one of them (C13-1) is the product of a cDNA truncated in the N terminus (Hata et al., 1991), so it is not possible to characterize sequence criteria to distinguish between a1 and a2. However, cydGm shows a quite different expression pattern from that of cyc3Gm and should therefore be classified as a novel class distinct from other A-type cyclins. Among these tentative classes, the soybean cyclins we have identified fall into three distinct classes, a1, a2, and [31, all of which are expressed in different meristematic organs of soybean plants. Several distinct cyclins identified from single plant species, such as maize (CydaZm, CydbZm, Cyc2Zm, and 1151 Cyc3Zm) and Arabidopsis (CydAt, Cyc2aAt, Cyc2bAt, CycSaAt, and Cyc3bAt), also fall into different classes. Therefore, these classes are suggested not only to reflect the genetic divergence between plant species but also to represent classes of cyclins that have different functions in the plant cell cycle. The previous observations for cell cycle-specific expression of a few of these plant cyclins also support this explanation. Two Antirrhinum cyclins, cyclAm and cyc2Am, which would belong to class (31, showed an expression pattern essentially the same as that of cycSGm (Fobert et al., 1994). Furthermore, cycSaAt and cycSbAt of Arabidopsis, which would belong to class 0.2, have been suggested to be expressed earlier in the cell cycle than cyc2aAt and cyc2bAt, which would belong to class (32 (Ferreira et al., 1994). The results presented in this study clearly demonstrate that the transcription of distinct classes of mitotic cyclin genes is regulated differentially in the plant cell cycle. These genes are Figure 7. Double-Labeling of Shoot Apices with cydGm and Histone H4, and with cydGm and cycSGm. The arrows indicate the positions of cells with hybridization signals from both probes. (A) and (B) Detection of hybridization signals with the digoxigenin-labeled cydGm probe (red) followed by that of the fluorescein-labeled histone H4 probe (black in [B]). (C) and (D) Detection of hybridization signals with the digoxigenin-labeled cydGm probe (red) followed by that of the fluorescein-labeled cycSGm probe (black in [D]). Bar = 25 urn. The Plant Cell 1152 expressed in all meristematic tissues at different times during the cell cycle, suggesting that they each have essential and distinct functions in cell cycle progression in plant meristems. Recently, Ferreira et al. (1994) have reported the presence of an organ-specific cyclin in Arabidopsis. They have shown that cyc2bAt mRNAs are present only in roots, whereas another closely related gene, cyc2aAt, is expressed in all plant organs; both of them would be classified as B-type (p2) cyclins by our criteria. Furthermore, two closely related cdc2 genes, cdc2S5 and cdc2-S6, have been suggested to be expressed differentially in various organs of soybean (Miao et al., 1993). S f 4k Our study showed no significant specificity of expression of the five distinct soybean cyclins in different meristematic organs and tissues. However, sequencing of the PCR products revealed the presence of a number of different molecular species of cyclin mRNAs, particularly A-type cyclins (data not shown). Because our hybridization analysis might not be appropriate to distinguish such individual mRNA species belonging to the same group, use of 3' noncoding regions as probes and/or of reverse transcription-PCR with primers specific to individual sequences may be necessary. It would be intriguing to analyze, in greater detail, the differential expression of *< '*. *. . Y -*•> " » • f **• ,^ ^»m *11 •*• J3WW B Vt* t t Figure 8. In Situ Hybridization of Shoot Apical Regions with 35S-Labeled Probes of Histone H4, cycSGm, and cyc3Gm. The hybridization signals are visible as black dots. Metaphase (m) and early telophase (t) cells can be seen. Sections were poststained with toluidme blue. (A) Hybridization with histone H4. Bar = 20 urn. (B) and (C) Hybridization with cyc5Gm. Metaphase and early telophase cells exhibit positive signals. Apparently nondividing cells also exhibit strong signals (indicated by arrowheads). Bars = 20 urn. (D) to (F) Hybridization with cyc3Gm. Arrowheads indicate cells with positive signals. Bars = 10 urn. Differential Expression of Plant Cyclins a1 Cyc2Gm cyc3aAt Cyc3bAt Cyc3Gm Cyc2Zm a2 = Cyc2aAt Cyc2bAt fl al., 1989). Oligo(dT)-primed double-strandedcDNAs were synthesized from 2 to 5 pg of poly(A)+RNA using a cDNA synthesis kit (Pharmacia Biotechnology), and cDNA libraries were constructed in hgtlO phages, as described by Kouchi and Hata (1993). Genomic DNA was obtained from etiolated hypocotyls of 7-day-oldsoybean seedlings, according to the method of Rogers and Bendich (1988). Polymerase Chain Reaction and Cloning of Cyclin cDNAs Cyc4Gm CycSGm CyclAt Cycl aZm Cycl bZm 1153 Pl P2 Cyc2Ms Cyc3Zm Figure 9. Phylogenetic Tree of Mitotic Cyclins from Higher Plants. The tree was depicted from a sequence alignment of the cyclin box indicated in Figure 1, according to the method of Higgins and Sharp (1988). Length of the horizontal lines reflects divergence. The sources of published sequences of plant cyclins are as follows: C131 from carrot (Hata et al., 1991); CycSaAt, Cyc3bAt, CycSaAt, Cyc2bAt, and CyclAt from Arabidopsis (Hemerly et al., 1992; Ferreira et al., 1994); CyclAm and Cyc2Am from Antirrhinum(Fobert et al., 1994); CyclaZm, CyclbZm, CycPZm, and Cyc3Zm from maize (Renaudin et al., 1994); Cyc2Ms from alfalfa (Hirt et al., 1992). The five soybean cyclins described in this paper are indicated in boldface. these cyclin genes in different organs or tissues andlor in response to environmental stimuli, such as infection with Rhizobium. Such analysis may provide more insights into how these cell cycle-related genes are involved in plant developmental programs. Degenerated oligonucleotide primers were designed for highly conserved regions of published amino acid sequences of mitotic cyclins. Their sequences are 5’ATI(C/T)Tl~lGATTGG(C/T)TIGTI(G/C)A(A/G)GT-3‘ (sense) and 5’-TCTGG(T/G)GG(A/G)TA(G/C)AT(C/T)TC(C/T)TCATATTT-3‘ (antisense), representing amino acid sequences of ILVDWLV(E/Q)V and KYEE(I/M)YPP(D/E), respectively (Hata et al., 1991). Deoxyinosinetriphosphate(designatedI in the primer sequence) was used to decrease the degree of degeneration. The polymerase chain reaction (PCR) amplification was performedwith oligo(dT)-primed cDNA prepared from poly(A)+ RNAs of young, developing soybean root nodules and root tips using Tth DNA polymerase (Toyobo, Tokyo). The primer concentrationswere 2 pM, and the cycling conditions were 30 cycles at 94°C (1 min), 45% (1.5 min), and 72% (2 min). The amplified fragments (185 bp) were isolated from the agarose gel, cloned into a pBluescript II SK+ plasmid vector (Stratagene), and sequenced. The selected PCR products were labeled by random priming with 32P-dCTPand used to screen the cDNA libraries of 9-day-old nodules and of suspension-cultured cells by standard procedures (Sambrook et al., 1989). Hybridization was carried out in 50% formamide, 4.8 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M trisodium citrate, pH 7.0), 5 x Denhardt’s solution (1 x Denhardt’s solution is 0.020/0 Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.5% SDS, 50 mM Hepes, pH 7.3, and 100 pglmL denatured salmon sperm DNA at 42% for >16 hr. The filter was washed in 2 x SSC, 0.5% SDS for 30 min at room temperature, and then in 0.5 x SSC and 0.1% SDS at 55OC four times for 10 min each. lnserts of the purified phages were subcloned into pBluescript II SK+ plasmids for further characterization. RNA and DNA Transfer Blot Analysis Agarose gel electrophoresis of total RNA and digested genomic DNA was performed using standard procedures (Sambrook et al., 1989). The RNA and DNA in the gel were blotted onto nitrocellulose membranes and hybridized with 32P-labeledcDNA inserts. The conditions of hybridization and subsequent washings were the same as those given for the screening of cDNA libraries. METHODS DNA Sequencing Plant Growth and Library Construction Soybean (Glycine max var Akisengoku) seedlings were grown with or without inoculation with Bfadyrhizobium japonicum, as described by Kouchi and Hata (1993). Suspension-culturedcells of soybean, initiated from hypocotyls of seedlings in the presence of 2,4-dichlorophenoxyacetic acid, were cultured in 85 medium (Gamborg, 1975) for 2 years. Total RNA was isolated from the tissues or cells by guanidium thiocyanate extraction, followed by centrifugation on cesium chloride, and the poly(A)+RNA-enriched fraction was selected by oligo(dT)-cellulose column chromatography by standard procedures (Sambrook et The cDNA inserts subcloned into pBluescript plasmids were sequenced by the dideoxy chain termination method (Sanger et al., 1977) using an automatic sequencer (Model37OA; Applied Biosystems,Foster City, CA). 60th strands were entirely sequenced. In Situ Hybridization Preparation of digoxigenin-labeled antisense RNA probes was as described by Kouchi and Hata (1993). The RNA probes corresponding to entire cDNA sequences (poly[A] tails were removed if present) were 1154 The Plant Cell synthesized using T3 or T7 RNA polymerase (Stratagene) and employed after partia1hydrolysis with sodium carbonate. The sense probes were also prepared and used as negative controls. Shoot apices of 7-day-old soybean seedlingswere fixed in 4% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 10 mM sodium phosphate buffer, pH 7.2, containing 100 mM NaCI, dehydrated through a graded ethanol series, and embedded in paraffin wax as described by Kouchi and Hata (1993). The sections (&pm thick) were attached to poly-L-lysine-coated slides. In situ hybridization procedures were the same as those described by Kouchi and Hata (1993), except for a minor modification in which washes with 2 x SSC and 1 x SSC containing 50% formamide were simply replaced by those with 2 x SSC and 0.5 x SSC without formamide, respectively. The method of double-target in situ hybridization was essentially the same as the method developed by Fobert et al. (1994). Fluoresceinlabeled RNA probes were prepared using the same protocol as that given for the preparation of digoxigenin-labeledprobes using fluorescein-11-rUTP (Boehringer Mannheim) instead of digoxigenin-11-rUTP. The sections were hybridized with a mixture of two probes; one was labeledwith digoxigenin and the other with fluorescein. After hybridization, the digoxigenin-labeledprobe was detectedwith anti-digoxigeninalkaline phosphatase in combination with Fast Red TRlnaphtol AS-MX (Sigma). The slides were mountedwith glycerin, and microphotcgraphs were taken. The coverslips were then removed, and the slides were incubated in 2 x SSC at 68OC for 3 hr to inactivate the alkaline phosphatase. The second, fluorescein-labeled probe was detected with the anti-fluorescein-alkaline phosphatase conjugate (Boehringer Mannheim; 1:lOOO diluted) in combinationwith nitro blue tetrazolium salt/5-brom~-chloro-3-indolyl phosphate toluidinium salt (Kouchi and Hata, 1993). Then the slides were mounted with glycerin, and photographs were taken. In some cases, the slides were further dehydrated through a graded ethanol series and mounted with Eukitt (O. Kindler, Germany). This step enabled us to confirm the overlappingof signals from digoxigenin- and fluorescein-labeled probes in the same cell, because the precipitation from Fast Red in the first round of detection diminished completely in ethanol. The in situ hybridization procedure with 35S-labeledRNA probes was as described by van de Wiel et al. (1990). The sections were counterstained with toluidine blue to visualize dividing cells. ACKNOWLEDGMENTS We thank Miki Maeda, Norimitsu Hariguchi, and TakuoYamamotofor their technical assistance. We also thank Drs. Wei-Cai Yang and Ton Bisseling for their suggestions and help with the in situ hybridization experiments with 3%-labeled probes and for providing the soybean histone H4 cDNA. This research was supported by a grant-in-aid(No. BMP-IV-95-1-1)to H.K.from the Ministry of Agriculture, Forestry, and Fisheries of Japan. Received March 21, 1995; accepted June 21, 1995. is differentiallyexpressed in leaves, protoplasts and during various cell cycle phases. Plant MOI. Biol. 20, 1121-1130. Colasanti, J., l'yers, M., and'sundaresan, V. (1991). lsolation and characterization of cDNA clones encoding a functional p34OdC* homologuefrom Zea mays Proc. Natl. Acad. Sci. USA 88,3377-3381. Evans, T., Rosenthal, E.T., Youngbloom, J., Dlstel, D., and Hunt, T. (1983). Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cell division. Cell33, 389-396. Feiler, H.S., and Jacobs, T.W. (1991). Cloning of the pea cdc2 homologue by efficient immunologicalscreening of PCR products. Plant MOI. Biol. 17, 321-333. Ferreira, P.C.G., Hemerley, A.S., Vlllaroel, R., Van Montagu, M., and Inzb, D. (1991). The Arabidoposis functional homolog of the ~ 3 4 ~ 0 ~ ~ protein kinase. Plant Cell 3, 531-540. Ferreira, P., Hemerly, A., Engler, J.D., Bergounioux, C., Burssens, S., Van Montagu, M., Engler, G., and Inzb, D. (1994). Three discrete classes of Arabidopsis cyclins are expressed during different intervalsof the cell cycle. Proc. Natl. Acad. Sci. USA91, 11313-11317. Fobert, P.R., Coen, E.S., Murphy, G.J.P., and Doonan, J.H. (1994). Patternsof cell division revealed by transcriptionalregulation of genes during the cell cycle in plants. EMBO J. 13, 616-624. Gamborg, O.L. (1975).Callus and cell culture. In Plant Tissue Culture Methods, O.L. Gamborg and L.R. Wetter, eds (Saskatoon: National Research Councilof Canada, Prairie Regional Laboratory),pp.1-10. Girard, F., Strausfeld, U., Fernandez, A., and Lamb, N.J.C. (1991). Cyclin A is required for the onset of DNA replication in mammalian fibroblast. Cell 67, 1169-1179. Hashimoto, J., Hirabayashi, T., Hayano, Y., Hata, S., Ohashi, Y., Suzuka, I., Utsugi, T., Tohe, A., and Kikuchi, Y. (1991). lsolation and characterizationof cDNA clones encoding cdc2 homologues from Oryza sativa-A functional homologue and cognate variants. MOI. Gen. Genet. 233, 10-16. Hata, S. (1991).cDNA cloning of a nove1 cdc2+/CDC28-related protein kinase from rice. FEBS Lett. 279, 149-152. Hata, S., Kouchi, H., Suzuka, I., and Ishii, T. (1991). lsolation and characterization of cDNA clones for plant cyclins. EMBO J. 10, 2681-2688. Hemerly, A., Bergounioux, C., Van Montagu, M., Inzk, D., and Ferreira, P. (1992). Genes regulating the plant cell cycle: lsolation of a mitotic-likecyclin from Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 89, 3295-3299. Higgins, D.G., and Sharp, P.M. (1988). CLUSTAL: A package for performing multiple sequence alignment on a microcomputer. Gene 73, 237-244. Hirayama, T., Imajuku, Y., Anai, T., Matsui, M., and Oka, A. (1991). ldentification of two cell-cycle-controlling cdc2 gene homologs in Arabidopsis thaliana. Gene 105, 159-165. Hirt, H., PBy, A., Gyorgyey, J., Bak6, L., Nkmeth, K., Borge, L., Schweyen, R.J., Heberle-Bors, E., and Dudits, D. (1991). Complementation of a yeast cell cycle mutant with an alfalfa cDNA ~ ~ Natl. ~ Acad. ~ . encoding a protein kinase homologous to ~ 3 4Proc. Sci. USA 88, 1636-1640. REFERENCES Hirt, H., Mink, M., Pfosser, M., Bogre, L., Gyorgyey, J., Jonak, C., Gartner, A., Dudits, D., and Heberle-Bors, E. (1992). Alfalfa cyclins: Differentialexpression during the cell cycle and in plant organs. Plant Cell 4, 1531-1538. Bergounioux, C., Perrenes, C., Hemerly, A.S., Qin, L.X., Sarda, C., Inzk, D., and Gadal, P. (1992). A cdc2 gene of Petunia hybrida Hirt, H., Phy, A., Bogre, L., Meskiene, I., and Heberle-Bors, E. (1993). cdc2Ms6, a cognate cdc2 gene from alfalfa, complements the Gl/S Differential Expression of Plant Cyclins 1155 but not the G2/M transition of budding yeast cdc28 mutants. Plant J. 4, 61-69. Reed, S.I. (1991). G, specific cyclins: In search of an S-phase promot- Hunt, T. (1991). Cyclins and their partners; from a simple idea to com- Renaudin, J.P., Colagnti, J., Rime, H., Vban, Z.A., and Sundaresan, V. (1994). Cloning of four cyclins from maize indicates that higher plicated reality. Semin. Cell Biol. 2, 213-222. Jacobs, T. (1992). Control of the cell cycle. Dev. Biol. 153, 1-15. Klng, R.W., Jackson, P.K., and Kirschner, M.W. (1994). Mitosis in transition. Cell 79, 563-571. ing factor. Trends Genet. 7, 95-99. plants have three structurally distinct groups of mitotic cyclins. Proc. Natl. Acad. Sci. USA 91, 735-7379, Rogers, S.O., and Bendich, A.J. (1988). Extraction of DNAfrom plant nodulin cDNAs representinggenes expressed at early stages of soybean nodule development. MOI. Gen. Genet. 238, 106-119. tissues. In Plant Molecular Biology Manual, S.B. Gelvin, R.A. Schilperoort,and D.P.S. Verma, eds (Dordrecht: Kluwer Academic Publishers), pp. A6/1-A6/11. Kühne, C., and Linder, P. (1993). A new pair of B-type cyclins from Sambmok, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Clon- Kouchi, H., and Hata, S. (1993).lsolation and characterization of novel Sacchammycescerevisiae that function early in the cal1cycle. EMBO J. 12, 343-3447. ing: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press). Lehner, C.F., and OFarrell, P. (1990). Expression and function of Dro- Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing sophila cyclin A during embryonic cell cycle progression. Cell 56, 957-968. with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Lew, D.J., Dulic, V., and Reed, S.I. (1991). lsolation of three novel Schwob, E., and Nasmyth, K. (1993). CLB5 and CLB6, a new pair human cyclins by rescue of G, cyclin (Cln) function in yeast. Cell 66, 1197-1206. of B cyclins involved in DNA replication in Sacchammycescerevisiae. Genes Dev. 7, 1160-1175. Miao, G.-H., Hong, 2.-L., and Verma, D.P.S. (1993). Two functional Sherr, C.J. (1993). Mammalian G1-cyclins. Cell 73, 1059-1065. Sherr, C.J. (1994). G1 phase progression: Cycling on cue. Cell 79, soybean genes encoding ~ 3 4 protein ~ ~ ~kinases ' are regulated by different plant developmental pathways. Proc. Natl. Acad. Sci. USA 90, 943-947. Minshull, J., Blow, J.J., and Hunt, T. (1989).Translation of cyclin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 56, 947-956. Minshull, J., Golsteyn, R., Hill, C.S., and Hunt, T. (1990).The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 9, 2865-2875. Nakayama, T., and Iwabuchi, M. (1993). Regulation of wheat histone gene expression. Crit. Rev. Plant Sci. 12, 97-110. Nash, R., Tokiwa, G., Anand, S., Erickson, K., and Futcher, A.B. (1988). The W H W gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J. 7,4335-4346. Norbury, C., and Nurse, P. (1992). Animal cell cycles and their control. Annu. Rev. Biochem. 61, 441-470. Nurse, P. (1994). Ordering S phase and M phase in the cell cycle. Cell 79, 547-550. Pagano, M., Pepperkik, R., Verde, F., Ansorge, W., and Draetta, G. (1992). Cyclin A is requiredat two points in the human cell cycle. EMBO J. 11, 961-971. 551-555. Soni, R., Carmichael, J.P., Shah, Z.H., and Murray, J.A.H. (1995). A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. Plant Cell 7, 85-103. Surana, U., Amon, A., Dowzer, C., Mcgrew, J., Byers, B., and Nasmyth, K. (1993). Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J. 12, 1969-1978. Swenson, K.I., Farrell, K.M., and Ruderman, J.V. (1986). The clam embryo protein cyclin A inducesentry into M phase and the resumption of meiosis in Xenopus oocytes. Cell 47, 861-870. van de Wiel, C., Scheres, B., Franssen, H.J., Van Liemp, M.J., van Kammen, A., and Blsseling, T. (1990).The early nodulin transcript ENOD2 is located in the parenchyma (inner cortex) of pea and soybean root nodules. EMBO J. 9, 1-7. van't Hof, J. (1985). Control points within the cell cycle. In The Cell Division Cycle in Plants, J.A. Bryant and D. Francis, eds(Cambridge: Cambridge University Press), pp. 1-13. Peeper, D.S., Parker, L.L., Ewen, M.E., Toebes, M., Hall, F.L., Xu, M., Zantema, A., van der Eb, A.J., and Piwnica-Worms, H. (1993). Yang, W.-C., de Blank, C., Meskiene, I., Hirt, H., Bakker, J., van Kammen, A., Franssen, H., and Bisseling, T. (1994). Rhizobium A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes. EMBO J. 12, 1947-1954. Nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation. Plant Cell 6, 1415-1426. Pines, J. (1993).Cyclins and cyclin-dependentkinases-Take your partners. Trends Biochem. Sci. 18, 195-197. Pines, J., and Hunter, T. (1990). Human cyclin A is adenovirus ElA- Zindy, F., Lamas, E., Chenivesse, X., Sobczak, J., Wang, J., Fesquet, D., Henglein, B., and Brechot, C. (1992). Cyclin A is required in associated protein P60 and behaves differently from cyclin B. Nature 346, 760-763. S phase in normal epithelial cells. Biochem. Biophys. Res. Commun. 182, 1144-1154. Distinct classes of mitotic cyclins are differentially expressed in the soybean shoot apex during the cell cycle. H Kouchi, M Sekine and S Hata Plant Cell 1995;7;1143-1155 DOI 10.1105/tpc.7.8.1143 This information is current as of June 17, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298 X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY