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American Society of Plant Biologists Genome-Wide Analysis of Core Cell Cycle Genes in Arabidopsis
a Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 1 To whom correspondence should be addressed. E-mail diinz{at}gengenp.rug.ac.be; fax 32-9-2645349
Cyclin-dependent kinases and cyclins regulate with the help of different interacting proteins the progression through the eukaryotic cell cycle. A high-quality, homology-based annotation protocol was applied to determine the core cell cycle genes in the recently completed Arabidopsis genome sequence. In total, 61 genes were identified belonging to seven selected families of cell cycle regulators, for which 30 are new or corrections of the existing annotation. A new class of putative cell cycle regulators was found that probably are competitors of E2F/DP transcription factors, which mediate the G1-to-S progression. In addition, the existing nomenclature for cell cycle genes of Arabidopsis was updated, and the physical positions of all genes were compared with segmentally duplicated blocks in the genome, showing that 22 core cell cycle genes emerged through block duplications. This genome-wide analysis illustrates the complexity of the plant cell cycle machinery and provides a tool for elucidating the function of new family members in the future.
Cell proliferation is controlled by a universally conserved molecular machinery in which the core key players are Ser/Thr kinases, known as cyclin-dependent kinases (CDKs). CDK activity is regulated in a complex manner, including phosphorylation/dephosphorylation by specific kinases/phosphatases and association with regulatory proteins. Although many cell cycle genes of plants have been identified in the last decade (for review, see Stals and Inzé, 2001  ), the correct number of CDKs, cyclins, and interacting proteins with a role in cell cycle control is unknown. Now that the complete sequence of the nuclear genome of Arabidopsis is available (Arabidopsis Genome Initiative, 2000), it is possible to scan an entire plant genome for all of these core cell cycle genes and determine their numbers, positions on the chromosomes, and phylogenetic relationships. From an evolutionary point of view, this core cell cycle gene catalog would be extremely interesting because it allows us to determine which processes are specific to plants and which are conserved among all eukaryotes. Furthermore, there is a unique opportunity to unravel in future experiments the functions and interactions of newly found family members of primary cell cycle regulators, thus expanding our knowledge of how the cell cycle is regulated in plants.
Nevertheless, a genome-wide inventory of all core cell cycle genes is possible only when the available raw sequence data are annotated correctly. Although genome-wide annotations of organisms sequenced by large consortia have produced huge amounts of information that benefits the scientific community, this automated high-throughput annotation is far from optimal (Devos and Valencia, 2001 Generally, annotation is performed in two steps: first, structural annotation, which aims to find and characterize biologically relevant elements within the raw sequence (such as exons and translation starts); and second, functional annotation, in which biological information is attributed to the gene or its elements. Unfortunately, there are some problems inherent to both.
When structural annotation is performed, the first problem occurs when no cDNA or expressed sequence tag (EST) information is available, which is the case for 60% of all Arabidopsis genes (Arabidopsis Genome Initiative, 2000 The problem with functional annotation is related to the difficulty of linking biological knowledge to a gene. Such a link is made generally on the basis of sequence similarity that is derived either from full-length sequence comparisons or by means of multiple alignments, patterns, and domain searches. Of major concern is the origin of the assigned function, because the transfer of low-quality or faulty functional annotation information propagates incorrect annotations in the public databases. Even correct annotations can be disseminated erroneously: one can easily imagine the transfer of a good functional assignment from a multidomain protein to a protein that has only one of the domains. This problem can be avoided using only experimentally derived information to predict unambiguously a gene's structure and function. Here, we applied a homology-based annotation using experimental references to build a full catalog with 61 core cell cycle genes of Arabidopsis. In total, 30 genes are either new or genes for which the previous annotation was incorrect. Based on phylogenetic analysis, we updated and rationalized their nomenclature. Furthermore, relations between gene family members were correlated with large segmental duplications.
Strategy To correctly annotate all core cell cycle genes, a strategy was defined that uses as much reliable information as possible, combining experimentally derived data with the best prediction tools available for Arabidopsis (see Methods). First, experimental representatives for each family were used as bait to locate regions of interest on the different chromosomes. For these selected regions, genes were predicted and candidate genes were validated; the presence of mandatory domains in their gene products was determined by aligning them with the experimental representatives; if necessary, the predicted gene structure was modified using the family-related characteristics or ESTs. In some cases, however, this approach did not allow us to conclude whether a region of interest really coded for a potential gene or whether a candidate gene was a core cell cycle gene. To clarify such situations, a more integrated analysis was performed. First, the members of every family were used to build a profile for that specific family. By taking the new predicted genes into account when creating the profile, a more "flexible" (i.e., all diversity within a class/subclass being represented) and plant-specific profile could be established. With this new profile, novel family members were sought within a collection of genome-wide predicted Arabidopsis proteins. Subsequently, the predicted gene products were again validated or modified by comparing them with those of other family members in a multiple alignment. With this additional approach, we could determine clearly whether the predicted genes were similar to a certain class of cell cycle genes.
To characterize subclasses within the gene families, phylogenetic trees were generated that included reference cell cycle genes from other plants and known genes from Arabidopsis. By different methods and statistical analysis of nodes, the significance of the derived classification was tested. Based on the position on the tree and the presence of class-specific signatures, genes were named according to the proposed nomenclature rules for cell cycle genes (Renaudin et al., 1996
Annotation and Nomenclature CDK In yeast, one CDK is sufficient to drive cells through all cell cycle phases, whereas multicellular organisms evolved to use a family of related CDKs, all with specific functions. In plants, two major classes of CDKs, known as A-type and B-type CDKs, have been studied to date. The A-type CDKs regulate both the G1-to-S and G2-to-M transitions, whereas the B-type CDKs seem to control the G2-to-M checkpoint only (Hemerly et al., 1995 ; Magyar et al., 1997; Porceddu et al., 2001). In addition, the presence of C-type CDKs and CDK-activating kinases (CAKs) have been reported (Magyar et al., 1997; Umeda et al., 1998; Joubès et al., 2001). Whereas the latter were shown to regulate the activity of the A-type CDKs, the function of the C-type CDKs remains unknown. To date, one A-type and four B-type CDKs have been described for Arabidopsis (Joubès et al., 2000; Boudolf et al., 2001). Furthermore, C-type CDKs and one CAK have been reported as well (Umeda et al., 1998; Lessard et al., 1999). In alfalfa, one E-type CDK has been identified, but no counterparts had been found previously in Arabidopsis (Magyar et al., 1997). By the homology-based annotation method used here, we identified eight CDKs (one A type, four B type, two C type, and one E type) and four CAKs (three D type and one F type).
The previously described CAK homolog of Arabidopsis (cak1At) differs substantially from the known rice CAK, R2 (Umeda et al., 1998
Cyclins Monomeric CDKs have no kinase activity and must associate with regulatory proteins called cyclins to be activated. Because cyclin protein levels fluctuate in the cell cycle, cyclins are the major factors that determine the timing of CDK activation. Cyclins can be grouped into mitotic cyclins (designated A- and B-type cyclins in higher eukaryotes and CLBs in budding yeast) and G1-specific cyclins (designated D-type cyclins in mammals and CLNs in budding yeast). H-type cyclins regulate the activity of the CAKs. All four types of cyclins known in plants were identified, mostly by analogy to their human counterparts. For Arabidopsis, at present, four A-type, five B-type, five D-type, but no H-type cyclins have been described (Soni et al., 1995 ; Renaudin et al., 1996; De Veylder et al., 1999; Swaminathan et al., 2000). Using the known plant cyclin sequences as probes, a total of 30 cyclins were detected in the Arabidopsis genome. For 19 cyclins, an EST was found (Table 1).
Three different subclasses of plant A-type cyclins (A1, A2, and A3) have been described (Renaudin et al., 1996
B-type cyclins are subdivided into two subclasses, B1 and B2. In total, Arabidopsis contains nine B-type cyclins, of which four belong to the B1 class (CYCB1;1, CYB1;2, CYCB1;3, and CYCB1;4) and four belong to the B2 class (CYCB2;1, CYCB2;2, CYCB2;3, and CYCB2;4). One gene could not be attributed to either the B1 or the B2 class, although it clearly contained a B-type–like cyclin box in combination with the B-type–specific HxKF signature. On the other hand, no B1- or B2-like destruction box was detected. The phylogenetic position of this gene within the B cluster depended on the number of positions used for the analysis. Because cyclin sequences are known to be saturated with substitutions (Renaudin et al., 1996
In addition to the five D-type cyclins described previously (CYCD1;1, CYCD2;1, CYCD3;1, CYCD3;2, and CYCD4;1), five new D-type genes were detected. Based on their phylogenetic positions, two of these genes were assigned to the D3 class (CYCD3;3 and CYCD3;4) and one was assigned to the D4 class (CYCD4;2). The remaining new D-type cyclins were subdivided further into classes CYCD5, CYCD6, and CYCD7 according to their phylogenetic positions. It is remarkable that CYCD4;2 and CYCD6;1 do not contain the LxCxE retinoblastoma (Rb) binding motif, whereas CYCD5;1 contains a divergent Rb binding motif (FxCxE) located at the N terminus. The biological functions of cyclins lacking the conserved Rb binding motif remain unclear. One Arabidopsis gene was found with high sequence similarity to cyclin H of poplar (71%) and rice (66%). Aligning all cyclins allowed us to identify the cyclin and destruction box consensus sequences for A-, B-, D-, and H-type cyclins (Table 2). Although A- and B-type cyclin boxes are very similar, these two types of cyclins can be discriminated by their destruction boxes. For two genes within the A- and B-type cyclins (CYCA3;1 and CYCB3;1), no destruction box could be detected. In addition, these genes have a highly diverged cyclin box compared with their subclass consensus. The low overall sequence similarity within D-type cyclins also is reflected in their cyclin boxes.
In addition to the cyclins described above, two presumed pseudogenes were predicted that were very similar to B-type cyclins. The precise number of pseudogenes for the seven selected families remains unclear, because the detection of pseudogenes depends on the degree of conservation in the gene structure and the degree of detection by prediction tools of these degenerated structures.
CDK/Cyclin Interactors and Regulatory Proteins
Upon the occurrence of stress or the perception of antiproliferation agents, the CDK/cyclin complexes are repressed by the CDK inhibitor (CKI) proteins. In mammals, two different classes of CKIs exist (the INK4 and the Kip/Cip families), each with its own CDK binding specificity and protein structure. Seven CKI genes belonging to the group of Kip/Cip CKIs have been described previously for Arabidopsis, designated KRP1 to KRP7 (De Veylder et al., 2001 ). No extra KRPs were detected in the complete genome, and no plant counterparts of the INK4 family were found.
CDK/cyclin activity is regulated negatively by phosphorylation of the CDK subunit by the WEE1 kinase and positively when the inhibitory phosphate groups are removed by the CDC25 phosphatase. A single WEE1 gene was identified on chromosome 1. The WEE1 kinase was annotated using two cDNA sequences that were at our disposal (L. De Veylder, unpublished results) and has its highest homology with the WEE1 kinase of maize, showing 56% similarity to the gene product of a partial mRNA (Sun et al., 1999
Rb and E2F/DP
The third group contains three new genes with an internal similarity of 59% and a sequence similarity with both E2F (21%) and DP genes (18%), initially indicating some kind of relation with the E2F/DP genes. When the boxes present in the E2F genes (DNA binding, dimerization, Marked, and Rb binding boxes) and the DP genes (DNA binding and dimerization boxes) were compared with those in the three new genes, only a DNA binding domain was found, but in duplicate (Figure 5A) . Both DNA binding domains are highly similar to the E2F DNA binding domain. Because of their phylogenetic positions, they form a distinct class, which we designated DP-E2F–like (DEL). The DNA binding domains of the E2F and DP genes have a limited across-family homology (Figure 5B), including the RRxYD DNA recognition motif (in their  3-helices), which interacts with half of the palindromic promoter binding site (CGCGCG and CGCGCG). Within all three DEL genes, the conserved DNA recognition motif RRxYD also is present in two copies. The E2F/DP heterodimer binds and recognizes the palindromic sequence of the binding site in an essentially symmetric arrangement (Zheng et al., 1999). Protein secondary structure prediction for the DEL genes showed that the winged-helix DNA binding motif, a fold found in the cell cycle transcription factors E2F/DP (three -helices and a  -sheet), is present in duplicate in all of these DEL genes. The first and second DEL DNA binding domains have an overall similarity of 61 and 47%, respectively, with the E2F DNA binding domain. Currently, no experimental data are available regarding the putative function and role of the DEL genes in cell cycle regulation.
Gene/Genome Organization To determine whether the segmental or genomic duplications and the acquisition of new cell cycle regulation mechanisms are linked, we mapped all cell cycle genes on the five different chromosomes (Figure 6) . Subsequently, all duplicated regions in the Arabidopsis genome were defined, and the position of every cell cycle gene was compared with the coordinates of each duplicated block.
Comparison of the positions of A2 cyclin genes with the positions of duplicated blocks in the Arabidopsis genome revealed that all four members are located in duplicated blocks: one internal duplication on chromosome 1 (CYCA2;3 linked with CYCA2;4) and one on chromosome 5 (CYCA2;2 linked with CYCA2;1). The three CYCA3 genes are organized in tandem (CYCA3;2, CYCA3;3, and CYCA3;4 spanning a region of <8 kb) and have a highly similar gene structure (number and size of exons and introns) as well as highly similar protein sequences (74.3% overall similarity). Only CYCA3;2 had one significant EST hit, whereas CYCA3;4 had an additional small predicted exon (33 nucleotides) compared with the other CYCA3 genes that occur in the same tandem (Figure 3B). Like the A2-type cyclins, all four B2-type cyclins were located within duplicated blocks: one duplicated block between chromosomes 2 and 4 (linking CYCB2;1 and CYCB2;2) and one internal duplication on chromosome 1 (linking CYCB2;3 and CYCB2;4). Although 10 D-type cyclins were detected in total, few of them were located in duplicated blocks. CYCD3;2 and CYCD3;3 are members of an inverted block between chromosomes 5 and 3, whereas CYCD4;1 and CYCD4;2 are located within an internal block of chromosome 5. The two CKS genes were located in a gene tandem duplication in which the stop codon of CKS2 was separated by only 916 bp from the start codon of CKS1 (Figure 3A).
Special attention is required for two duplication events. On chromosome 1, a large internal duplication occurred (spanning an area of Another special, internally duplicated event was found on chromosome 5. Two duplicated blocks (Figure 5, brown blocks) were detected that connected both extremities of the chromosome. Although these blocks could be regarded as one, we clearly distinguished an invertedly duplicated block between them (Figure 6, blue block). CYCD4;1 and CYCD4;2 both fit nicely into the first block. CDKC;1 and CDKC;2 mapped to this region as well, located in the small invertedly duplicated block. It is remarkable that, although both pairs of linked genes were located in duplicated blocks with different orientations, their relative positions were the same (i.e., at the bottom and at the top of chromosome 5, a C-type CDK was followed by a D4-type cyclin). This configuration suggests that, initially, one large duplication event occurred (Figure 6, the region spanning the brown and blue blocks) that was reshuffled later by inversions (and perhaps some deletions), resulting in adjacent, duplicated blocks with different orientations and sizes.
The members of the Arabidopsis genome sequencing consortia use different tools to perform automated genome annotations and determine similarities to ESTs and known protein sequences to refine gene models. This procedure has generated a large quantity of information on the Arabidopsis genome. However, the extraction of clear biological information for a particular process from these public databases is not always easy (for instance, the word "cyclin" as a query in the Martiensried Institute for Protein Sequences database returned 37 hits, with 23 putative cyclin or cyclin-like hits). To solve this problem, we designed a protocol focused mainly on high-quality, homology-based annotation. We used a combination of two selected high-quality Arabidopsis prediction tools (Pavy et al., 1999 ; Schiex et al., 2001; C. Mathé and P. Rouzé, personal communication) together with pure experimental information as reference material. One advantage of this method is that the chance of finding new and rarely expressed genes is maximized, because they are structurally characterized by tools with higher specificity and sensitivity than those used by the different consortia to generate genome annotations (Gopal et al., 2001). Second, the focus on families with available experimental references allows comparisons with functionally well-characterized genes and diminishes the risk of the propagation of incorrect annotations. In addition, the use of hidden Markov profiles, which represent the complete diversity within a family, clearly is more powerful than the use of a single sequence for remote homolog detection (Karplus et al., 1998).
With this strategy, we have built a catalog of 61 core cell cycle genes belonging to seven selected families. From these, 30 genes had not been described before, and for 22 of them the gene prediction provided by the Arabidopsis Genome Initiative (2000)
Like mammals, plants evolved to use different classes of CDKs to regulate their cell cycle. In Arabidopsis, six different CDK classes can be identified, designated A through F. Although some of these CDKs have been proven to be active during specific phases of the cell cycle (Magyar et al., 1997
Within the CDK family, we identified three new CAK members that are close homologs of the rice R2 gene (Hata, 1991
The complexity of the cyclin gene family appears to be higher in plants than in mammals. Compared with human, Arabidopsis has
In contrast to the A- and B-type cyclins, D-type cyclins lack high sequence similarity to each other, which is reflected in the phylogenetic analysis resulting in seven D-type subclasses. Compared with A- and B-type cyclins, of which some complete subclasses (A2 and B2) are located within segmentally duplicated blocks, no large duplications can be found for the D-type cyclins. Only the D3 and D4 subclasses have different members. Redundancy of the D3-type cyclins has been proposed previously as an explanation for the failure to observe mutant phenotypes when knocking out a single D3-type cyclin (Swaminathan et al., 2000
The much larger divergence seen for D-type cyclins compared with A- and B-type cyclins might reflect the presumed role of D-type cyclins in integrating developmental signals and environmental cues into the cell cycle. For example, D3-type cyclins have been shown to respond to plant hormones, such as cytokinins and brassinosteroids, whereas CYCD2 and CYCD4 are activated earlier in G1 and react to sugar availability (for review, see Stals and Inzé, 2001
Although plants clearly share all of the elements needed for G1/S entry with other higher eukaryotes, they lack the typical class of E-type cyclins, which are known to be essential regulators of DNA replication (Duronio et al., 1996
It is surprising that mammals and plants have approximately the same number of core cell cycle genes, with the exception of the difference in cyclin number described above. Complex, multicellular organisms may need many more cell cycle genes to coordinate cell cycle progression with their diverse developmental pathways. However, the pool of mammalian cell cycle genes probably is larger than expected because of the frequent occurrence of alternative splicing. For example, spliced variants of cyclin E are known, with an expression profile and substrate specificity different from those of cyclin E itself (Mumberg et al., 1997 E2F/DP transcription factors are characterized by the presence of both a DNA binding domain and a transcription activation domain. Binding of these transcription factors to the E2F/DP palindromic binding site is mediated by a small DNA recognition motif (RRxYD). By scanning the genome for E2F/DP-related proteins, a putatively novel class of cell cycle–regulating genes was identified, designated DEL. The DEL proteins have two E2F-like DNA binding boxes, each including the RRxYD motif, but they have no activation domain. By competing for the same DNA binding sites, monomeric DEL proteins could act as competitors of the E2F/DP proteins, and because they lack an activation domain, they would act as repressors of E2F/DP-regulated genes. This mechanism would avoid the G1-to-S transition in cases in which conditions are not appropriate for entry into the S phase (such as DNA damage and stress). This new class of putative cell cycle regulators seems not to be plant specific, because one homolog was found in Caenorhabditis elegans (data not shown). In conclusion, our genome-wide analysis demonstrated an unexpected complexity of the core cell cycle machinery in plants that is comparable with that seen in mammals. The major challenge for the future is to understand the specific role of these individual genes in regulating cell division during plant development.
Annotation of Arabidopsis thaliana Cell Cycle Genes The genome version of January 18, 2001 (version 180101), was downloaded from the ftp site (ftp://ftpmips.gsf.de/cress/) of the Martiensried Institute for Protein Sequences (Martiensried, Germany). Regions of interest on the chromosomes were localized with BLAST software (Altschul et al., 1997 ) with experimental representatives as query sequences. For the regions returned by BLAST, chromosome sequences were extracted with 15 kb upstream and downstream from the hit to prevent unreliable predictions caused by border effects.
Gene prediction was performed with EuGene (Schiex et al., 2001
The predicted candidate gene products were aligned with the experimental representatives using CLUSTAL W (Thompson et al., 1994
Phylogeny and Nomenclature
Protein Structure Analysis
Segmental Duplications in the Arabidopsis Genome
Accession Numbers
NOTE ADDED IN PROOF
We especially thank Yvan Saeys for providing us with the necessary programs to define duplicated blocks in the Arabidopsis genome, Dr. Yves Van de Peer for help with the analysis of saturated positions in the cyclin alignments, Dr. Catherine Mathé for additional information regarding EuGene, Patrice Déhais for the programs developed to run EuGene, Sébastien Aubourg for helpful discussions, and Martine De Cock and Rebecca Verbanck for help in preparing the manuscript and artwork, respectively. This work was supported by grants from the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister's Office—Federal Office for Scientific, Technical and Cultural Affairs; P4/15), the European Union (ECCO QLG2-CT1999-00454), Génoplante (Project BI1999087), and CropDesign N.V. (0235). K.V. is indebted to the Vlaams Instituut voor de Bevordering van het Wetenschappelijk–Technologisch Onderzoek in de Industrie for a predoctoral fellowship, and L.D.V. is a postdoctoral fellow of the Fund for Scientific Research (Flanders).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010445. Received October 11, 2001; accepted January 23, 2002.
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Abstract
INTRODUCTION
4890 kb, or 16% of chromosome 1) that was followed by several inversions (data not shown), leading to the formation of multiple smaller blocks, one of which contained two pairs of cell cycle genes: CDKB2;2 linked with CDKB2;1 and CYCB2;3 linked with CYCB2;4. The CYCB2;3 gene was present in tandem (interspersed by one gene), and the second copy was designated Arath; CYCB2;3–pseudo, because its gene structure was degraded and imperfect with respect to CYCB2;3. We conclude that this tandem duplication occurred after the segmental duplication event, because in the region linked to the duplicated block, no trace of another extra B2-like cyclin was found. 
