Core cell cycle regulatory genes in rice and their expression profiles across the growth

zone of the leaf

A. Pettkó-Szandtner​1,3*​, M. Cserháti​1,2,3*​, R.M. Barrôco​3,5*​, D. Dudits​1​, G.T.S. Beemster​3,4

1​
Biological Research Center, HAS, Temesvári krt 62, H-6726 Szeged, Hungary
2​
Nebraska Medical Center, Omaha, NE, 68198-5145
3​
Plant Systems Biology, VIB, Technologiepark 927, B-9052 Zwijnaarde, Belgium
4​
Department of Biology, University of Antwerp
5​
CropDesign N.V./BASF, Technologiepark 921C, 9052 Ghent (Zwijnaarde), Belgium

*​
R.M. Barrôco, M. Cserháti and A. Pettkó-Szandtner are equal contributors.

Corresponding author:
Aladár Pettkó-Szandtner
Email:​ ​pettko-szandtner.aladar@brc.mta.hu

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Abstract

Rice (​Oryza sativa L.) as a model and crop plant with a sequenced genome offers an
outstanding experimental system for discovering and functionally analyzing the major cell
cycle control elements in a cereal species. In this study, we identified the core cell cycle genes
in the rice genome through a Hidden Markov Model (HMM) search and multiple alignments
supported with the use of short protein sequence probes. In total we present 55 rice putative
cell cycle genes with locus identity, chromosomal location, and approximate chromosome
position and EST accession number. These cell cycle genes include 9 CDK genes, 27 cyclin
genes, 1 CKS gene, 2 RBR genes, 9 E2F/DP/DEL genes, 6 KRP genes, and 1 WEE gene. We
also provide characteristic protein sequence signatures encoded by CDK and cyclin gene
variants. Promoter analysis by the FootPrinter program discovered several motifs in the
regulatory region of the core cell cycle genes. As a first step towards functional
characterization we performed transcript analysis by RT-PCR to determine gene specific
variation in transcript levels along the rice leaves. The meristematic zone of the leaves where
cells are actively dividing was identified based on the cell length profile and flow cytometry.
As expected, expression of the majority of cell cycle genes was exclusively associated with
the meristematic region. However genes such as d​ ifferent ​D-type cyclins​, DEL1, KRP1/3, and
RBR2 were also expressed in leaf segments representing the transition zone in which cells
start differentiation.

Keywords
Rice, cell cycle, CDK, cyclin, leaf growth zone, gene annotation, transcript analysis

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INTRODUCTION

Rice is unique among cereals being a staple crop, on which half of the human population
depends as its main source of nutrition (Calingacion et al. 2014), and on the other hand an
experimental model species fully amenable to functional genomics studies (Xu et al. 2005). In
particular, rice possesses a relatively small diploid genome (430 Mb), has a relatively short
generation time and facilitates efficient Agrobacterium​-mediated transformation (​Ozawa,
2009​). Furthermore, the rice genome shows considerable co-linearity or synteny with the
genomes of other cereals, such as wheat, barley, and maize (Keller and Feuillet 2000). These
similarities warrant that results in rice research can be expanded to other cereal crops (Xu et
al. 2005). Grasses are now frequently treated as a single genetic system, allowing the transfer
of biological information from a well-studied model grass genome, such that of rice, to related
crop species (Huang et al. 2013).
With the entire rice genome sequence available, considering its importance as a model crop
and given the central role of the cell cycle machinery in plant growth and development, it is
essential to characterize its core cell cycle machinery (Guo et al. 2007). A similar
genome-wide study of another rice gene family for genes encoding SET domain proteins
involved in histone methylation based on sequence analysis and global microarray profiling
(Lu et al. 2013) provides a solid basis for further research into the function of that gene family
in plant development.
CDKs and their activating subunits, cyclins, are the basic component units of the core cell
cycle machinery. Sequential formation and activation of specific CDK/cyclin complexes
controls the progression through the cell cycle. The activity of CDK/cyclin complexes relies
on several regulatory mechanisms, including phosphorylation, regulation of protein stability,
as well as the interaction with other regulatory proteins. Also in plants, considerable progress
has been made in unraveling the basic mechanisms controlling cell cycle progression, and the
understanding of plant cell cycle machinery has been reviewed extensively (Dewitte and
Murray 2003; Gutierrez 2005; Inzé 2005; Dante et al. 2014; Desvoyes et al. 2014). Two types
of CDKs (​CKDA and CDKB​) and three groups of cyclins (​CYCA, CYCB and CYCD)​ are well
known to control the cell cycle in plants. CDKs are regulated positively by CDK Activating
Kinases (CAKs). Two types of CAKs have been identified in plants (CDKD and CDKF), and

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one of them itself forms a CDK/CYC complex (CDKD/CYCH). The activity of mitotic CDKs
is universally inhibited by phosphorylation in the proximity of the active region. Plants
possess homologues of the WEE type kinases responsible for this phosphorylation, but no
homologs of the CDC25 phosphatase, which in other systems reverses the inhibition. Earlier a
CDC25-like phosphatase has been identified in Arabidopsis,​ suggesting that plants also have a
phosphatase that can activate CDK/CYC complexes, although the precise role of this
CDC25-like protein still remains unclear as (reviewed by Inzé, 2005 and Francis, 2011).
A further level of regulation of CDK activity involves cyclin-dependent kinase inhibitors
(CKIs). CKI proteins directly inhibit CDK activity by binding to the CDK/CYC complexes
(reviewed by Verkest et al., 2005). In plants, all CKI proteins that have been identified share a
limited similarity to the mammalian p27​Kip1 inhibitor, based on which plant CDK inhibitors
were designated Kip-related proteins (KRPs, De Veylder et al. 2001). In contrast to the
mammalian inhibitors, the conserved CDK/CYC binding domain is located at the
carboxy-terminal side of the protein.
The heterodimeric E2F/DP factors mediate the transcription of many S-phase specific genes
being negatively controlled by pocket proteins such as retinoblastoma proteins (RB; reviewed
by Gutierrez, 2005. and ​Kuwabara ​and ​Gruissem​, 2014). ​The function of plant RBR proteins
is under the control of phosphorylation by kinases and phosphatases mediating stress and
developmental signals (reviewed by Dudits et al., 2011). In contrast to the unicellular
eukaryote yeast, plants and animals utilize the RB/E2F pathway for cell cycle regulation,
which suggests that this pathway is closely associated with the differentiation and
development of multicellular organisms. However, it was demonstrated that the unicellular
algae Chlamydomonas reinhardtii and Ostreococcus tauri also possess RB homologues,
suggesting the existence of an RB regulatory pathway in those eukaryotes as well (Bisova et
al. 2005; Robbens et al. 2005).
In rice as well as other grass species, growing leaves expand predominantly lengthwise.
Growth of leaves in monocotyledonous species, such as rice, results from cell proliferation at
the basis of the leaf and on cell expansion that takes place in the elongation-only zone,
directly above the meristem (Beemster et al., 1996; Rymen et al., 2007). The rate of leaf
elongation is determined by the cell production in the meristem and rate and duration of cell
expansion in the elongation zone, both located at the base of the leaf and enclosed by the
sheaths of older leaves.

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In the present study we characterize the core rice cell cycle genes using informatics tools and
we position them in phylogenetic trees. We also present functional information by
characterizing their expression pattern along the leaf growth zone to gain insight in their
involvement in division and differentiation events in leaves.

RESULTS

Identification of Cell Cycle Genes in the Rice Genome

To identify the genes of the rice core cell cycle machinery, we scanned the rice genome using
an HMM search. Multiple alignments were performed for each (sub) class of cell cycle genes
by using available sequence information of known cell cycle genes from monocot and dicot
species. We also used short (<50 aa) protein sequence probes taken from individual protein
sequences representing the different cell cycle gene families. These were used in an HMM
search to interrogate the rice DNA sequence database. In total 55 rice putative cell cycle genes
were identified, and divided into the following groups: 9 CDK genes, 27 cyclin genes, 1 CKS
gene, 2 RBR genes, 9 E2F/DP genes, 6 KRP genes, and 1 WEE gene (for a more precise
distribution and characterization of those genes among subclasses see Table 1). Noteworthy is
that we identified no B3-type cyclin gene.

Phylogeny and Nomenclature

Phylogenetic trees were constructed for the CDK A and B gene families, as well as for the
cyclin D, the E2F/DP/DEL, and RB families, respectively. To assign gene names, In the
presee nomenclature previously outlined for Arabidopsis (Vandepoele et al. 2002). Some of
the genes referred here (mostly the cyclins) and corresponding proteins have already been
named before. In that case, we adopted as much of the previously proposed nomenclature as
possible. In the case of the cyclin family a number of contradictions were found between the
cyclins names proposed by Wang et al. (2004) and La et al. (2006), and Guo et al. (2007).
Therefore in these cases we gave preference to the names proposed by La et al. (2006) as they
are the most comprehensive. Two genes were not identified before, and we named them in
accordance with their relationships with other members of the same class and respective
location in the phylogenetic clades (see Table 1).

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Cyclin-dependent kinases
CDKs are Ser/Thr kinases, which play a key role in cell cycle regulation. The most widely
conserved CDKs possess a canonical PSTAIRE motif. In yeast, a single PSTAIRE CDK is
sufficient to regulate all phases of the cell cycle. In contrast, plants encode several types of
CDKs with functions in cell cycle regulation, reviewed by Dudits et al. (2007). The plant
PSTAIRE CDK, designated as CDKA, can functionally replace its yeast orthologs,
cdc2/CDC28 (Colasanti et al. 1991; Ferreira et al. 1991; Hirt et al. 1991). Plant-specific
B-type CDKs have been found in several plants, and are specifically expressed in the
G2-to-M phases (Magyar et al. 1997; Porceddu et al. 2001). D- and F-type CDKs are
CDK-activating kinases (CAK) that serve to activate A-type CDKs (Umeda 2005). CDKD
binds cyclin H to form a CAK complex, while CDKF is a plant-specific CAK, which seems to
phosphorylate CDKD, thereby creating an important phosphorylation cascade (Yamaguchi et
al. 2000; Umeda et al. 2005). Plant C-type CDKs have been implicated in transcriptional
control (Barrôco et al. 2003). No function has yet been determined for E-type CDKs in plants
(Magyar et al. 1997).
Based on the present search, the rice genome encodes orthologs for each of the known
plant-type CDKs (A to E). The number of rice genes (9) coding for CDK proteins is
essentially similar to that of Arabidopsis (12). Interestingly, rice possesses 3 A-type CDKs
(one in​ Arabidopsis)​ but only two B-type ones (4 in​ Arabidopsis​; Figures 1 and 2).
One CDKD was found with the NFTALRE signature, and one CDKE gene with the
SPITAIRE signature.
Phylogenetic analysis revealed that two of the A-type CDKs are very closely related, whereas
the third member clusters apart, therefore we have named rice CDKA genes as CDKA1;​ 1
(previously cdc2Os1​), CDKA1​;2 and CDKA2;​ 1 (previously cdc2Os2​). Based on characteristic
protein signatures common to rice CDK genes (Table 2), we distinguish at least two CDKA
subclasses, which seems to be in common with other grass species, such as diploid wild
einkorn wheat (​Triticum urartu​) (Ling, 2013) and maize (Rymen et al. 2007). As far as the
B-type rice CDKs are concerned, we could categorize two subclasses CDKB1 and CDKB2
similarly to other plant species (Figure 2). ​It is a general trend that CDKBs as well as CDKAs
are​ ​both​ ​present in monocots and dicots.
Cyclins

6
Cyclins are the activating units of CDKs, and formation of cyclin-CDK complexes is essential
for kinase activity. In plants and animals, there are three main classes of cyclins shown to
regulate the cell cycle (A-type, B-type, and D-type cyclins). The A and B cyclin groups are
highly conserved in eukaryotes, while the D-type cyclins are divergent between animals and
plants (Wang et al. 2004). However, the D-cyclins of plants and animals share some structural
similarities, such as a conserved N-terminal LXCXE motif. The LXCXE peptide binds to the
pocket region of RB-related proteins (RBRs) targeting them for CDK phosphorylation.
A total of nearly 50 putative cyclins were previously identified in Arabidopsis and in tomato
(Vandepoele 2002, et al. Wang et al. 2004, Zhang et al. 2013). Based on phylogenetic analysis
several classes were defined (A, B, C, D, H, L, T, and U), but only a number of A-type,
B-type, and D-type cyclins have been functionally characterized to date. From those, A-type
cyclins were shown to be implicated in the S-to-G2 and G2-to-M transitions; cyclins from
group B are essential for M-phase entry; D-type cyclins drive cells through the G1-to-S (Inzé
2005). A number of plant D-type cyclins might also be implicated in driving G2-to-M
transition (Mészáros et al. 2000; Schnittger et al. 2002; Kono et al. 2003; Koroleva et al.
2004; Pettkó-Szandtner et al. 2006). Hu et al. (2010) identified 59 cyclins in the maize
genome, distributed among 10 chromosomes, which were grouped into six types by
phylogenetic analysis.
We identified 27 rice cyclins belonging to class A-, B-type and D-type, and one cyclin H.
Most cyclins identified have already been described previously (Wang et al. 2004; La et al.
2006, Guo et al. 2007), but the successive release of later rice genome versions, together with
a number of inaccuracies and inconsistencies identified in (and between) those works justifies
an updated compilation of those rice cyclins involved in cell cycle regulation. All three works
have additionally identified a number of rice cyclins described as F- (unique to rice), L-, P- T-
and U-type, but their function as true cell cycle regulatory genes remains to be demonstrated.
For the cyclin A-group, the individual subclasses A1, A2, and A3 clustered separately. The
cyclin A1 genes cluster into separate monocot and dicot clades, but the same does not apply to
the other A-type cyclins. Similarly, B1- and B2-type rice cyclins clearly cluster separately.
From the B-type cyclins 4 putative CYCB1 and 2 CYCB2 proteins were identified. In the B1
cyclin group, dicots and monocots cluster clearly separate into two separated clades (data not
shown). For the A-type and B-type cyclins, typical sequence signatures were detected, such as
the cyclin box, the LVEVAEEY consensus (with variations), and the H(Y/H)(R/K)(L/F)

7
motif. Possible destruction boxes were also investigated. PEST sequences were not identified
in A- or B-type cyclins, with the exception of CYCB2;1. Typical TS(V/T)LTARSKAAC
sequence signatures were found in B1-type cyclins, and KYEE motifs were found in 4 A-type
and 4 B-type cyclins. Additionally, B-type cyclins contain an NLS signal in their N-terminal
part (Table 2).
From the three classes analyzed here, the D-type cyclins formed by far the largest group with
14 identified genes, subdivided into six subclasses (3 CYCD1, 1 CYCD3, 5 CYCD4, 3
CYCD5, 1 CYCD6, and 1 CYCD7). Rice D-type cyclins present overall low sequence
similarity, but the phylogenetic analysis showed that the cyclin D2 and cyclin D4 family
members were not separated (Figure 3), suggesting that these two subfamilies are in fact part
of one large group (hereafter named as D4-type cyclins). For all cyclin D subfamilies, we
identified a p-destruction box and a cyclin box sequence. The typical RB-binding motif
LxCxE is absent in CYCD6;1. PEST motifs were found for 5 D-type cyclin genes. Typical
KMEE sequences were present in all cyclin D genes, except for cyclin D6 and D7. For cyclin
H only the cyclin box signature was found. Here as in the case of CDK genes ​all 6 types of
cyclin Ds form one group including monocot and dicot species, signaling the basic
importance of these genes to the core cell cycle machinery.

CDK inhibitors
Cyclin-dependent kinase inhibitors (CKIs) are important regulators of cell cycle progression
in higher eukaryotes. Despite the low sequence similarity with their mammalian counterparts,
plant CKI genes encode functional CKIs, as demonstrated by their ability to inhibit CDK
activity (Verkest et al. 2005; Meguro et al. 2014). All known plant CKIs share homology with
the N-terminal domain of the mammalian Kip family of CDK inhibitors, being therefore
named as Kip-related proteins (KRPs). ​Six ​KRPs were identified in the rice genome, sharing
characteristic motifs with seven previously described cyclin-dependent kinase inhibitors from
Arabidopsis thaliana​. Two of them, Orysa;KRP4 (Os10g33310) and Orysa;KRP1
(Os02g52480), contain a previously identified motif (​RT/ST/SRET/STPCSLI​), which is also
present in a number of dicots (Pettkó-Szandtner et al. 2006). Rice CKIs belong to two groups,
INK4 and Kip/Cip, where the gene family KRP belongs to the Kip/Cip family of genes in
rice. No INK4 inhibitors were found in rice.

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The 6th ​rice ​KRP gene (Orysa;KRP6) is extremely short and is not expressed in leaf or cell
suspension (data not shown). However because 9 ESTs were found in the NCBI database, 8 of
which came from a stress library, suggests that this KRP gene is not directly involved in cell
cycle control, but rather in stress response. This idea is supported by the fact that in
Arabidopsis the N-terminal region of the KRP1 gene, which is responsible for degradation, is
lacking in this rice gene (Schnittger et al. 2003). Apart from the expression studies described
here, some rice KRPs were further characterized revealing for example and important role of
KRP5 in rice seed formation (Barrôco et al. 2006; Yang et al. 2011).

E2F/DP and RBR

E2F transcription factors play a special and important role in driving the cell from G1 phase
to S phase. The E2F/DP/RBR pathway is conserved between mammals and plants, as well as
in some unicellular eukaryotes such as Chlamydomonas reinhardtii and Ostreococcus tauri,​
reviewed by Inzé (2005) and by Gutierrez (2005). A. thaliana contains three E2F (​a to c​), two
DP (​a and b​) and three DEL genes (​1 to 3)​ . E2Fs typically have a DNA binding, DP
heterodimerization, RBR interaction and transactivation domains. On the other hand, DELs
are atypical E2F proteins, which have a duplicated DNA-binding domain and bind to DNA
without dimerizing with DP. It revealed that in addition to genes involved in DNA replication
control, genes with a diverse set of other functions are also potential E2F targets (Vandepoele
et al. 2005), suggesting that E2Fs govern the expression of a much wider network of genes
than those controlling S-phase entry and progression. DELs themselves are also targets of
E2F, which repress entrance into the endoreduplication cycle (Komaki and Sugimoto, 2012).
In the rice genome 9 E2F/DP/DEL genes (4 E2F, 3 DP and 2 DEL) were identified, some of
which have been previously described (Kosugi 2002; Cooper 2003). Sequence analysis
(Figure 4) shows that one of the identified E2F proteins (E2F3) is closely related to the E2Fc
of Arabidopsis,​ whereas the other 3 are not directly related to the Arabidopsis E2Fa or E2Fb
proteins. This strongly suggests that the rice E2F3 is the functional ortholog of the
Arabidopsis E2Fc protein, which, opposed to E2F1 and E2F2, functions as a transcription
repressor of E2F-responsive genes (del Pozo 2002).
Whereas the genomes of dicot plants (including Arabidopsis,​ poplar, tobacco and pea) code
for a single RBR, the rice and maize genomes include two and three RBR orthologs,
respectively (Sabelli et al. 2005; Lendvai et al. 2007; Rymen et al. 2007). As in other grasses,

9
rice seems to have distinct types of RBR proteins, suggesting that the E2F/RBR pathway in
monocots comprises a degree of complexity more close to that of animal systems (Cobrinik,
2005; Sabelli et al. 2005).

Other CDK/cyclin regulatory and binding proteins

WEE genes down-regulate the activity of CDKs through phosphorylation. As in Arabidopsis
and maize we identified a single WEE gene in the rice genome. Also one single rice CKS gene
was identified, contrary to Arabidopsis and maize in which a second CKS paralog was
identified (Vandepoele et al. 2002; Rymen et al, 2007).

Promoter analysis
Promoter analysis was performed on different gene groups where the promoter sequence was
available. The program FootPrinter (Blanchette and Tompa 2003) was used and
parameterized as described in the Materials and Methods section, allowing us to find motifs
for each individual core cell cycle gene in the rice genome (Table 3).
These motifs were then verified by checking them against motifs found in the PLACE
database (Higo et al. 1999). The most frequently occurring motifs -314MOTIFZMSBE1,
AGL2ATCONSENSUS, AGL3ATCONSENSUS, ATRICHPSPETE, MARTBOX,
CARGCW8GAT, and CARGNCAT from the PLACE database matched 3, 3, 3, 4, 5, 10, and
10 of the motifs predicted in Table 3, respectively. For example, the AGL2 protein which has
a predicted binding site in the genes CYCD4;1, CYCD4;2, CYCD4;5, CYCD5;2, and RB1​.
AGL2 interacts with OsMADS13 in developing ovules (Favaro et al. 2003). This motif along
with AGL3ATCONSENSUS, CARGCW8GAT, and CARGNCAT are all MADS-boxes,
which are conserved all across the plant kingdom (Ferrario 2004). MADS genes are
well-known to take part in plant development, when cells are actively dividing and progress
through the cell cycle. The motifs CARGCW8GAT and CARGNCAT occur in the promoters
of the following genes: CDKD;1, CYCB1;1-4, CYCD4;2, CYCD4;4, CYCD5;1, CYCD5;2,
KRP2,​ and​ DP3.​

Expression of rice cell cycle genes in the rice leaf

Characteristic distribution of cell division and expansion activity in monocotyledonous leaves

offers an ideal experimental objective for studies on expression patterns of core cell cycle

10
genes. Flow histograms resulting from the analysis of consecutive segments (1 cm) from one
representative leaf and 4C fraction distribution throughout the leaf shows that cell division
activity is restricted to the basal 2 cm of the leaf and that no endoreduplication occurs in this
species (Figure 5). ​The lengths of the cells along the leaf blade follow a characteristic
sigmoidal pattern from the base to the mature zone, where the transition from division to
expansion and from expansion to mature cells are located around 2 and 4 cm from the base,
respectively (Figure 5C). ​Note that a fraction of the mature cells is in the 4C state, although
they no longer divide or endoreduplicate. It is not possible to tell if these 4C cells are arrested
in G2 phase, or in the G1 of the first round of endoreduplication; ​one cannot discriminate this
(only 8C cells can be shown unambiguously to be endoreduplicating).

Based on the above information we used equivalent leaf segments for RNA isolation and
analysis of the relative expression level for these core cell cycle genes by quantitative
RT-PCR with gene-specific primers. The expression levels were first normalised for
differences between samples using geNorm (see Math and Meth). To allow for comparison
of the expression profiles for all cell cycle genes, irrespective of their expression levels, the
values for each gene were normalized to the lowest expression value along the leaf(Figure 6).
In order to asses the absolute exprsesion levels, we also plotted the maximum gene
expression level N​0​/∑N​0​*100 and relative fold change for all of the genes, ordered according
to relative fold change (Figure 6).

Cyclin-dependent kinases
The rice CDKB1;​ ​1 and CDKB2​;​1 transcript levels are relatively high in the 1 and 2 cm
segments corresponding to the meristem (Figure 6). In a similar fashion, CDKB1 has a high
basal expression in ​Arabidopsis,​ causing asymmetric cell division by forming a complex with
CYCD6;1 and targeting a phosphorylation pocket domain in RBR (Cruz-Ramírez, 2012).
CDKA1;​ ​1​, CDKA1;​ ​2 have comparable expression levels in the meristem. However, whereas
the A1 type genes have a constant expression level throughout the growth zone, the
expression level of CDKA2;1 is significantly lower and declines after the meristematic region
(Figure 6). The remaining CDKs ​have relatively strong expression with little fluctuanion
throughout the leaf (Figure 6.), similar to what was found in Arabidopsis (Beemster et al.

11
2005). In rice, CDKB1;1 and CDKB1;2 were shown to be highly expressed in the endosperm
(Fujita et al. 2010).

Cyclins
Within the rice cyclin gene family, the A- and B-type variants are differentially expressed
with relatively high expression in the meristematic region of leaves (Figure 6). In all cases we
can ​detect the expression peak in the first segment that gradually declines towards the tip
region. From the cyclin family CYCB1;​ ​4 is the cyclin with extremely high relative expression
level in the basal region of the leaf. The CYCA3;​ ​2 variant also has a high degree of
up-regulation in the meristem​. Two previously identified cyclins, CYCA3​;​3 and CYCA3​;​4​,
(Wang 2004) have not been assigned as members of the A1-, A2- or A3-subclasses as they
formed a separate clade outside of the A- and B-type cyclins, suggesting that they are not true
A-type cyclins. Based on expression of both of these genes and are most likely not involved
in cell proliferation. Contrary to the other A- and B-type cyclins, these two cyclin-like genes
were constitutively expressed along the growing leaf (data not shown). Also, a gene
previously identified as CYCB1;​ ​4 (La 2006) has been shown (based on Rice TIGR release 4)
to encode a putative mutator-like transposase (11972.t03759 = Os02g41720). We investigated
its expression and found that contrary to other B-type cyclins, this gene has a very high but
nearly constitutive expression in the rice leaf, being only weakly up-regulated (nearly 4x) in
the expansion region, contradicting its possible role as a mitotic cyclin (data not shown).
D-type cyclins show very diversified expression profiles with a lower differences along the
growth zone in comparison to cyclin A and B genes. CYCD5;3 is highly up-regulated in the 3
most basal leaf segments, whereas CYCD1;1, CYCD3;1, CYCD4;5 and CYCD6;1 transcripts
are very sparse in the meristem, and expressed mostly in the early expansion zone where cells
exit proliferation. The CYCD4;5 gene extends its expression up to the 4 cm segment where
differentiated cells are in the majority. Additionally, a set of D-type cyclins was found to be
constitutively expressed (CYCD1;3, CYCD4;4, CYCD5;1, CYCD7;1) (Figure 6.). They show
a similarity to the behavior of homologous genes in Arabidopsis (Beemster et al. 2005) and
maize (Rymen et al. 2007). Those cyclins are possibly regulated at other levels besides the
transcriptional level.
E2F/DP/DEL

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The levels of DEL transcripts are essentially modulated along the leaf growth zone (Figure 6).
The DEL1 gene is expressed in the first two leaf segments. We found preferential activity of
the DEL2 gene in the first two leaf segments, therefore this gene can be considered as a
regulatory component in dividing cells. In Arabidopsis​, the DEL1 gene is expressed mostly in
proliferating cells, acting as a transcriptional repressor of genes required for
endoreduplication (Berckmans et al. 2011). Because the rice leaf does not undergo
endoreduplication (Endo, 2012), a similar role cannot be hypothesized in these organs. As for
the rice DEL genes, the Arabidopsis DEL2 and DEL3 were also found to be expressed mostly
during the proliferation stages of leaf development or in the proliferating region of the root tip
(Beemster et al. 2005). However, it is also important to note that DEL1 gene from rice
(together with RBR2, KPR1/3 and CYCD4;5)​ forms a small group of cell cycle regulators with
a relatively high expression in segment 3 and 4 of the leaf, indicating a role for these genes in
the regulation of early differentiation or cell cycle exit.
We found that DPs are expressed nearly constitutive, DP3 is the most abundant DP. The
E2F2 gene showed an expression peak only in the first leaf segment representing the
meristematic region, comparable to the expression pattern in Arabidopsis and maize
(Beemster et al. 2005 and Rymen et al. 2007). The transcript level of E2F1 extends into the
expansion zone. ​E2F3​ and​ E2F4​ were expressed constitutively along the leaf (Figure 6).

CDK inhibitors
The KRP genes present distinct expression profiles with very different levels of transcript
accumulation. KRP5 is slightly up-regulated in the meristem, whereas KRP3 is a very
abundant gene that essentially peaks in the 3​rd and 4​th leaf segment coinciding with the exit
from the cell cycle. The ​KRP4 i​ s the second most abundant KRP, with moderate fluctuation,
wich peaks in the very end of leaf. Torres Acosta and co-workers (2011) have found similar
results. Conversely, KRP1 transcripts are present both in the proliferating and expanding
segments of the leaf, being slightly more abundant outside the dividing region. KRP2
transcript levels do not vary significantly along the leaf (Figure 6) .

RBRs
In the present study we detected essential differences in expression profiles of the RBR1 and
RBR2 genes. The RBR1 transcripts are extremely abundant in the 1​st segment of leaves with

13
high division activities (Figure 5, 6). Its relatively high expression drops extremely down in
the 2​nd segment of leaves. This profile may indicate a role for RBR1 in dividing cells. As far as
the RBR2 gene expression is concerned, we recorded higher levels throughout the leaf with a
moderate, but significant increase in segments 2 and 3. Previously, a similar trend was
reported, showing that the RBR1 gene is expressed in dividing rice cells while RBR2 is
preferentially expressed in differentiated cells (Lendvai et al. 2007). The expression of the
maize RBR2a, for which we did not identify a rice homolog​, was mitotic-specific in
developing endosperm and leaves and very different from that of RBR1,​ which is
constitutively expressed (Sabelli et al. 2005; Rymen et al. 2007).

Other CDK/cyclin regulatory and binding proteins

WEE1 expression is essentially up-regulated in the first leaf segment (nearly 30-fold),
suggesting that its expression is associated with dividing cells. Increased abundance of maize
WEE1 transcripts, during endosperm development, was found to coincide with the peak in
endoreduplication activity (Sun et al. 1999). In Arabidopsis​, the expression of WEE1
fluctuates during leaf development, being up-regulated mostly during proliferation stages as
in the maize leaf (Rymen et al. 2007), but is constitutive throughout different regions of the
root (Beemster 2005).
CKS1 h​ as a strong expression level which does not vary strongly throughout the leaf (Figure
6.). This expression profile is comparable mostly to that of the Arabidopsis and maize CKS1​,
as CKS2 expression is largely associated with cell proliferation, both in the leaf and root
tissues (Beemster et al. 2005; Rymen et al. 2007).
Most of the identified cell cycle genes are up-regulated in the first and second leaf segments,
covering the leaf meristematic region. A very small number of genes (DEL2, KRP1, and
RBR2) are moderately up-regulated in the initiation of the expansion zone, and an even
smaller number of genes have an irregular profile (Figure 6).
For about one-third of all genes (20) the expression levels do not reach 4-fold changes.
Combining the expression patterns of all analyzed genes shows the existence of essentially
four groups of genes. A first (largest) group is comprised of all genes that are largely
up-regulated in the leaf meristem, with the expression being the highest in the two most basal
leaf segments. Within this group, several genes (CYCB1;1, DEL2; RBR1) are extremely
up-regulated in the meristem relative to the differentiation zone. A second group of genes

14
(​CYCD1;1,​ ​CYCD3;1, RBR2, KRP1)​ , is not only expressed in dividing cells, but also in the
transition to differentiation. A third group of genes (​CYCD4;5, KRP3​) shows an increased
expression level beyond the division zone in the 3​rd leaf segment representing primarily
expansion function with an arrested cell cycle. In addition a last group of encompases nes that
are constitutively expressed throughout the leaf (e.g. most​ CDK​ and​ DP​ genes; see Table 5).

DISCUSSION

Identification of core cell cycle genes in various plant species is an essential prerequisite for
discovering the basic molecular events controlling organ growth and differentiation. The rapid
increase of DNA sequence information for the plant kingdom opened the way for
whole-genome searches of selected groups of genes. As a first example, the Arabidopsis
genome was screened and the major cell cycle control genes were identified (Vandepoele et
al. 2002; Menges et al. 2005). These studies have classified 99 cell cycle genes in
Arabidopsis.​ Guo et al. (2007) used the BLASTP program in NCBI and KOME on the
Arabidopsis key cell cycle proteins to identify core cell cycle genes in rice. This study
resulted in the discovery of 41 cell cycle genes. In the present work we extend this list by
analyzing a wider source of cell cycle genes based on an HMM search on the rice genome
sequence. Using a similar approach Rymen et al (2007) discovered 43 cell cycle genes in the
maize EST database. Overall in our annotation process, 55 rice cell cycle genes were found
(Table 1). In order to help in the annotation and correction process, rice ESTs were mapped to
the BAC clones found for every rice cell cycle gene. After annotation and careful inspection,
the genes were characterized and classified according to their own family and/or subfamily. In
order to do so, special sequence signatures were noted for every single gene family/subfamily.
Special signatures, destruction boxes, KYEE, and PEST sequences were also recorded for
each single gene.
Next, phylogenetic trees were constructed in order to position every gene within its own
family/subfamily. The number of members in the different cell cycle protein groups does not
differ significantly between rice and Arabidopsis​, or the other plant genomes where the
presence of cell cycle genes has been investigated genome-wide. Our data confirm the
previous conclusion by Guo et al. (2007) and Rymen et al. (2007) that the number of the core
cell cycle genes in rice and maize is lower than in the case of Arabidopsis.​ Considering that

15
the rice genome has been subject to large duplication events followed by extensive gene loss,
as previously proposed (Vandepoele et al. 2003), it is interesting to note that those events
seem not to have substantially affected the cell cycle gene machinery, as at first sight not
much divergence can be found between rice and​ Arabidopsis.​
The phylogenetic comparison of 30 A-type CDKs from more than 20 plant species (Figure 1)
showed that the clusters obtained strictly coincide with the taxonomic groups of the different
species, as previously reported (Joubès et al. 2000, Dudits et al. 2007). The existence in rice
and a few other species of more than one CDKA suggests that gene duplication events have
taken place within the different plant family lineages.
B-type CDKs form a gene family that is unique to plants, and its special sequence signature
varies between PPTALRE, PPTTLRE or PSTTLRE. This CDK group is found in 5 monocot
and dicot plant families, and in several unicellular algae, such as Ostreococcus​,
Chlamydomonas and Dunaliella.​ The only CDKB from O. tauri comprises a PSTALRE
motif, whereas in Chlamydomonas and Dunaliella CDKBs are characterized by the presence
of a PSTTLRE (Lin et al. 1999; Bisova et al. 2005; Robbens et al. 2005).
Both B-type CDKs from rice possess the PPTALRE hallmark; no CDK with a PPTTLRE
motif was found. The presence of the PPTALRE motif is normally characteristic of the
CDKB1 subclass, which would classify both those rice proteins as CDKB1-type. However,
Joubès et al. (2000) already pointed out a rice CDKB as being the most similar to the CDKB2
subclass. Indeed, after phylogenetic analysis, one of the B-type CDK sequences clusters with
the B1-type CDKs from several other species, whereas the other groups with their CDKB2
subclass, even though both hold the same signature motif (Figure 2). Sequence hallmarks
would therefore lead to the classification of this protein as a CDKB1,​ whereas sequence
homology points towards the same CDK as being a member of the​ CDKB2​ subgroup.
B-type CDKs show expression peaks as cells progress into G2 and M phase. Elevated
expression at the time of mitosis was described for several B-type CDKs (Magyar et al. 1997;
Menges et al. 2003; Menges and Murray, 2002, Zhiponova et al. 2006) and is consistent with
a role for CDKB in the regulation of the G2-to-M transition (Umeda et al. 1999a; Porceddu et
al. 2001). In Arabidopsis​, CDKB1 transcripts were shown to accumulate in late S phase/early
G2 and M phases, while CDKB2 expression was restricted to cells in mitosis (Endo et al.
2012). In accordance with these experimental findings the present expression profiling of
these CDKB genes in rice leaves clearly showed an active expression in the two lower

16
segments representing meristematic tissues with mitotically active cells (Figure 6). As
reviewed by Inagaki and Umeda (2011) internal and external signals can regulate CDK-cyclin
activity to modulate cell proliferation in specific developmental processes.
In the rice genome, two CDKC genes were identified, both containing the PITAIRE sequence
signature. Accumulation of CDKC transcripts does not correlate with cell division activity as
previously demonstrated for the Arabidopsis CDKC proteins (Barrôco et al. 2003). Also in
alfalfa, a C-type CDK was shown to be nearly constitutively expressed throughout the cell
cycle (Magyar et al. 1997, Fülöp et al. 2006).
As shown in Table 1, our present search revealed 27 rice cyclin genes, which is a low number
if we consider that La et al. (2006) predicted 48 rice cyclin genes distributed among 12
chromosomes. We failed to detect F-, SDS-, H-, L-, T- and P-type cyclins. The extended
recovery by La et al. (2006) may have resulted from the use of searching keywords as “human
cyclin” or “​Homo sapiens cyclin”. In the present work we used only plant sequences for the
HMM search.
Both monocot and dicot species have genes that belong to all CDK/cyclin/E2F/DEL/DP/RB
gene groups, suggesting that the D-type cyclin types diverged prior to the divergence of the
mono- dicot clades. Such genes are so conservative that they also appear in the single-celled
alga species, ​Chlamydomonas reinhardtii (Cross 2015). It is expected that core cell cycles
such as these perform a very basic function in the cell, explaining their ubiquitous distribution
in plants (Polyn, 2015).
Figure 6 shows that the activities of the A- and the B- type cyclin genes are the highest in the
meristematic leaf region. The transcript levels of the CYCB1;4 gene were especially high in
these two leaf segments and sharply reduced in the further segments. This gene strongly
responded to indole-3-acetic acid treatment (Guo et al. 2007). In comparison, IAA levels peak
at the base of the maize leaf growth zone (Nelissen et al. 2012).
The expression of some mitotic cyclins was investigated in rice roots revealing that CYCA1;​ ​1
expression peaks in G2, whereas transcripts of CYCB2;​ 1 and CYCB2;​ 2 accumulated in mitosis
(Umeda et al. 1999b). Several D-type cyclin genes (​CYCD1;3, CYCD4;4, CYCD7;1) a​ re
expressed at low levels and fluctuate moderately across the leaf growth zone. In Arabidopsis
the transcripts from the CYCD4;1 gene were detected in the shoot apical meristem, leaf
primordia and vascular tissues (Kono et al. 2003). We found three D-type cyclins (​CYCD1;1,
CYCD3;1, CYCD4;5,) to be expressed beyond the meristematic zone, representing the

17
transition towards differentiation. The observed expression profiles provide bases for detailed
characterization of the functional role of these cyclins.
In agreement with Lendvai et al. (2007), we found two types of RBR genes in rice. The
expression patterns confirmed that the RBR1 gene is exclusively active in meristematic region
of leaves while RBR2 gene has an almost constitutive expression in leaves, with a small but
significant increase at the exit of cell division zone (Figure 6). Nevertheless, even in the
meristem the expression levels of RBR2 are higher than RBR1, suggesting a dominant role in
leaf development. The maize RBR1 gene regulates CDK activity, the mitotic cell cycle,
endoreduplication, cell and nuclear sizes, and programmed cell death during the development
of the maize endosperm ​(Sabelli et al. 2013). The maize genome carries another pocket
domain RBR2a gene expressed in the mitotic stage during the developing endosperm (Sabelli
et al. 2005). Sequence similarities prompt us to suggest that RBR2 is the rice ortholog of
maize RBR2a,​ which in turn is closely related to the mammalian p107 RB-related protein.
Additional lines of evidences suggest that the maize RBR2a and the rice RBR2 are
functionally related to the mammalian p107 RB-like protein. Like the maize RBR2a,​ OsRBR2
but not OsRBR1​, also possess E2F regulatory binding sites in its promoter (Vandepoele 2005),
suggesting that expression of this gene is itself regulated by​ E2F/RB​.
Remarkable progress has been made in understanding the regulation mechanisms of the cell
cycle control in leaf development in dicot species, primarily with the model species
Arabidopsis thaliana. Studies on the growth control of cereal leaves have so far been much
less intensive (Gonzales et al. 2012; Kalve et al. 2014). Tardieu et al (2000) reported the
maximum in S-phase nuclei in the 0-20 mm zone beyond the leaf initiation point in maize,
where kinematic analyses showed cell division occurs (Ben-Hay-Salah et al. 1995; Rymen et
al. 2007; Nelissen et al. 2012) and where the highest CDK activity is located (Granier et al.
2000). Consistently in rice leaves the division activity was detected in the basal region of the
leaves (Figure 5).
In the leaf intercalary meristem, cells are produced in parallel cell files and move distally as a
result of continuous production of new cells. As the cells reach the elongation zone they stop
dividing, but keep expanding until they reach their final mature cell length. This spatial
gradient of cell division and cell expansion results in leaf growth taking place predominantly
along a one-dimensional axis. Due to this strict linear organization, the rice leaf presents an
excellent system to investigate the relationship between cell cycle control during organ

18
growth and cell cycle gene expression. The expression profiles of the core cell cycle gene can
serve as markers representing the actual cellular events. As shown in Figure 6, a set of
analyzed cell cycle genes were found to be active beyond the meristematic region. As
examples we can list the CYCD4;5, E2F1, KRP1/3, RBR1 genes expressed in the 3​rd segment
of leaves representing a transition zone between division and differentiation. The overall
expression patterns are different for these genes. The RBR1 and DEL2 genes show
exceptionally high up-regulation in the meristematic region. In Arabidopsis​, inhibitory
proteins, such as KRPs, are either constitutively expressed or endoreduplication-associated
(Beemster et al. 2005; Mizutani et al. 2010). Moreover, whereas KRPs are mostly
constitutively expressed in the Arabidopsis leaf, the accumulation of KRP transcripts in the 3​rd
segment of the rice leaf suggests an important role for some of these regulators during cell
proliferation arrest and may be related to the suppression of endoreduplication in this organ.
Overall, as most cell cycle genes from rice do not directly cluster together with members of
the corresponding families in Arabidopsis,​ it is not possible, based on sequence homology, to
establish possible functional orthologs between known Arabidopsis genes and rice. This, and
similar, transcriptional analysis of a large number of genes is therefore an important first step
in the efforts to functionally characterize rice cell cycle genes. The added value of this paper
is that we have been able to identify and characterize cell cycle genes in the regulation of leaf
growth, which has been lacking until now (Andriankaja, 2012; Polyn, 2015).

Conclusion
The rice genome sequence data provided a first-time prospect of unraveling cell cycle
regulation in this model cereal crop species. A fundamental first step in this effort was to
successfully identify the cell cycle genes and their respective expression patterns.
Here, we report the genome-wide survey and characterization of the rice core cell cycle genes.
Several of these cell cycle regulators have been found previously, but in most cases their
timing of expression was not linked to the developmental state of an organ. Our work not only
identifies novel cell cycle rice genes, but also provides a view of the transcriptional profile of
those genes in leaf tissues. These global analyses reveal that a large number of core cell cycle
genes are differentially expressed throughout the rice leaf, allowing us to form a more precise
view for the transcriptional regulation of the control of the rice cell cycle.

19
We show that the rice leaf is a powerful and interesting system to investigate cell cycle gene
expression in the context of organ formation. This work is a contribution to reaching a new
level of understanding of the mechanisms coordinating cell division with growth, during the
course of cereal organ development. The presented data can support future activities in
detailed functional characterization of key cell cycle genes in rice.

MATERIALS AND METHODS

Annotation, Phylogenetic Analysis and Tree Construction

Sequences coding for cell cycle proteins from monocot and dicot species (maize, barley,
wheat, rape, tobacco, tomato, sorghum, lucerne) were gathered by BLAST-ing against the
NCBI database. Cell cycle proteins from Arabidopsis were downloaded from
http://bioinformatics.psb.ugent.be/supplementary_data/klpoe/corecc/​. Multiple alignments
were created for each class or sub-class of cell cycle genes using clustalW. Afterwards, an
HMM profile was made which was used to scan the rice DNA sequences downloaded from
the TIGR database. A large number of sequences found by the HMM search were selected
(e-scores as great as 0.01). These sequences were multiple aligned using clustalW, and
sequences not identified as cell cycle genes were excluded. A second approach involved the
use of short (about 13-40 aa) protein sequence probes, which were used to sift through the rice
DNA sequence database, using the TBLASTN program. For this a data table was created to
record many attributes of the genes being annotated. More precisely, BAC accession numbers,
as well as positions on the chromosomes, EST numbers and typical sequence motifs were
recorded for each gene. The protein probes were also used to verify the results of the HMM
profile search. Overall in the annotation process, 55 rice cell cycle genes were found, while 35
of the genes had to be corrected, and 2 supposed pseudogenes were found. In order to help in
the annotation and correction process, rice ESTs were mapped to the BAC clones found for
every rice cell cycle gene. Another source of aid was the usage of similar cell cycle genes
found in​ Arabidopsis​, which were also mapped to the BAC clones.
After annotation and correction, the genes were characterized and classified according to their
own family and/or subfamily. This was accomplished by taking the following things into
consideration. First of all, special sequence signatures were noted for every single gene

20
family/subfamily. This was performed for the CDK and cyclin genes. Special signatures,
destruction boxes, KYEE, and PEST (poly-serine and threonine sequences which also serve
as destruction boxes) sequences were noted down for each gene. Next, phylogenetic trees
were constructed in order to help classify each and every single gene within its own
family/subfamily. The program used was the Create Tree program in the CLC Genomics
Workbench version 5.1. For this, multiple alignments of all protein sequences of the given
gene family were made with monocot and dicot species together. Bootstrapping was used for
making trees with 1000 samples per run.

Promoter analysis
Promoter analysis was performed for each gene family with the program FootPrinter
(Blanchette et al. 2003). For this, promoters from a number of other dicot and monocot plant
species were also extracted (​Arabidopsis thaliana​, Brassica oleracea,​ Lotus japonica​,
Medicago sativa​, Populus termulata,​ and Zea mays)​ in order to find maximally conserved
regulatory elements. Each promoter sequence was taken as 2 Kbp, but if it overlapped with
upstream genes, it was shortened. If the promoter was too short, then it wasn’t analyzed,
because the FootPrinter program needs sequences of a minimum length as input. If EST data
existed for a given gene, then the 5’ UTR was determined and also excluded from the
promoter region. Information for the promoter of each gene studied in the analysis can be
seen in the Supplementary Excel file.
FootPrinter was parameterized specially for each gene family. The parameters set included
motif length, maximum number of mismatches, subregion size (the size of the region within
which all motif instances occur), and triplet screening.

Growing of Rice Plants

Rice seeds (​Oryza sativa cv Nipponbare) were germinated in Jiffy peat pellets (Jiffy Products
International, Drøbak, Norway) and saturated with water. After 8 days, the plants (in the Jiffy
pellet) were transferred to 2L pots containing pond-soil. The plants were grown in a growth
room under high humidity and a short-day light regime. Growth room conditions were set to
60-70 % relative humidity, a temperature regime of 28°C day/22°C night, with a day length of
11 h.

21
Flow cytometry of rice leaves
The basal 100 mm of steady-state growing sixth leaves was cut into 1 cm segments. The
segments were chopped up with a razor blade in 2 mL ice-cold buffer (200 mM Tris-HCl, pH
7.5, 4 mM MgCl2, and 0.1% Triton X-100), to release the nuclei and debris filtered out using
a 30-mm mesh, stained with DAPI (De Veylder et al. 2001), and analyzed with a PARTEC
Cyflow analyzer. The relative abundance of nuclei with 2C and multiple DNA content using
FloMax software (version 2.4 d, PARTEC).

RNA Isolation and qPCR Analysis

Total RNA was isolated from leaves using RNeasy Plant Mini Kit (Qiagen GmbH, Hilden,
Germany). Five micrograms of each sample were reverse-transcribed into cDNA with the
SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA).
Quantitative RT-PCR amplification of the cDNA was carried out with the iCycler iQ
real-time PCR machine (Bio-rad, Hercules, CA, USA), with gene-specific primers designed
with the Beacon Designer 4.0 (Premier Biosoft International). The PCR reactions, run in
triplicate, were performed in 96-well optical reaction plates heated for 2 min to 95°C to
activate hot start Taq DNA polymerase, followed by 40 cycles of denaturation for 60 s at
95°C and annealing extension for 15 s at 60°C. PCR products were detected directly by
measuring the increase in fluorescence caused by the binding of SYBR Green I dye to
double-stranded DNA (Platinum SYBR Green qPCR SuperMix UDG; Invitrogen).
Fluorescence values were recorded during ​each cycle and represented the amount of product
amplified to that point in the amplification reaction. Each run was completed with a melting
curve analysis to confirm the specificity of amplification. C​t values were determined by the
iCycler software using a fluorescence threshold manually set to 150, for all runs, and exported
into MS Excel workbooks for analysis. Data analysis was based on the mathematical
functions described by Ramakers et al (2003), and Rutledge and Côté (2003). The
amplification efficiency (Eff) was calculated for each primer pair individually by determining
the slope of each individual PCR curve at its log-linear phase (Eff = 10​slope​). The individual C​t
values resulting from the triplicates were averaged and comparative template transcript
amounts (N​0​) were calculated as Eff​(-Ct)​. For each gene, changes in transcript levels were
determined by dividing the (N​0​) values of the seven samples by the smallest value. The
maximum gene expression level was calculated by dividing the genes transcript amounts (N​0​)

22
by the sum of all core cell cycle gene transcript amount. Expression analysis was also
performed on a number of rice genes that are commonly referred to as constitutively
expressed, such as actin1 (​ACT1​), polyubiquitin2 (​UBQ2)​ , sucrose phosphate synthase
(S​PSOs)​ and GOS2​, besides our set of cell cycle genes (many of which were shown to be
constitutively expressed).
Differences in the amount of target cDNA used for PCR were normalized with geNorm
(​http://medgen.ugent.be/~jvdesomp/genorm/​), using the most stably expressed genes for the
analyses (UBQ2; GOS2; KRP4; E2F3; E2F4; RBR2; CYCH1; CYCD5;1; CYCD1;3 CDKD;
CDKE; CDKC2; CDKC1; CDKA1;2; CDKA2;1), resulting in the selection of the following
seven genes for normalization (UBQ2; E2F4; CYCH1; CDKC1; CDKD; KRP1; CDKC2).
Due to the relatively high degree of variation in the results obtained from independent
experiments (biological replicas), we have mainly focused our analysis in expression changes
greater than 4–fold.

ACKNOWLEDGEMENTS
The authors would like to thank Wim Van Caeneghem for skillful advice with real-time PCR
analysis, Klaas Vandepoele for assistance in the annotation process of the rice cell cycle
genes, Yves van de Peer and the members of the Bioinformatics group for providing
appropriate infrastructure for the annotation process. This work was partly supported by a
grant from the Institute for the Promotion of Innovation by Science and Technology in
Flanders (postdoctoral fellowship to R.M.B.), A.P-Sz was supported by OTKA-68896 and the
János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

Declarations
We report no conflict of interest with anybody whatsoever. We declare that the work that we
have done is original, and has not been submitted to any other journal, and has not been
published anywhere else.

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Figure legends

Figure 1 – Neighbor-Joining tree of the CDKA proteins with the Poisson correction for
phylogenetic distance calculation. Bootstrap values of 1000 bootstrap iterations are shown.
Numbers indicate phylogenetic distance.

Figure 2 – Neighbor-Joining tree of the CDKB proteins with the Poisson correction for
phylogenetic distance calculation. Bootstrap values of 1000 bootstrap iterations are shown.
Numbers indicate phylogenetic distance.

Figure 3 – Neighbor-Joining tree of the CYCD proteins with the Poisson correction for
phylogenetic distance calculation. Bootstrap values of 1000 bootstrap iterations are shown.
Numbers indicate phylogenetic distance. The individual cyclin D subfamilies are shaded in
order to better distinguish between them.

Figure 4 – Neighbor-Joining tree of the E2F/DP/DEL/RB proteins with the Poisson correction
for phylogenetic distance calculation. Bootstrap values of 1000 bootstrap iterations are shown.
Numbers indicate phylogenetic distance. The individual gene families are circled with
different colors and labeled to better distinguish between them.

Figure 5 – Position of the meristematic region along the rice leaf. A: Flow histograms
resulting from the analysis of consecutive segments (1 cm) from a representative leaf. B. 4C
fraction distribution throughout the leaf. In all graphs vertical bars indicate SE.

Figure 6 – Expression profiles of rice cell cycle genes throughout the rice leaf, as determined
by qPCR analysis. The values represent expression fold change compared to the leaf segment
with the lowest expression. For each gene the segment is highlighted in red if its fold change
is higher than 1.75 times the average fold change and in orange if the expression level
fluctuates greater than 1-1.75 times than average. The leaf segments sampled cover three main
growth regions (as determined by kinematic analysis), which are depicted below the
expression table. The maximum ​absolute ​gene ​abundance value is noted in a separate column

33
to the left side of the table. The relative fold change is noted in a separate column on the right
side of the table. The genes are sorted according to​ ​decreasing relative fold change.

Tables
Table 1. Characteristics of 55 core cell cycle genes in​ Oryza sativa
Reference: Denotes whether gene was discovered in Wang et al. (2004) (W), La et al. (2006) (L), or Guo et
al. (2007) (G), or newly identified (NI)
Locus ID Proposed gene Previous Chr Strand Appr. pos. on EST accession No. of Referenc
name name . chromosome number exons e
CDKA
Os03g02680 Orysa;CDKA1;1 cdc2Os1 3 + 978000 dbjAK073961 8 G
Os03g01850 Orysa;CDKA1;2 3 + 490000 AU031081.1 8 NI
Os02g03060 Orysa;CDKA2;1 cdc2Os2 2 - 1180000 dbjAK063438 8 G
CDKB
Os01g67160 Orysa;CDKB1;1 1 - 38717000 C22366.1 7 G
Os08g40170 Orysa;CDKB2;1 cdc2Os3 8 + 24670000 CB659407.1 6 G
CDKC
Os05g 32360 Orysa;CDKC1;1 5 + 17689000 C74901.1 12 G
Os01g72790 Orysa;CDKC1;2 1 + 41867000 dbjAK103469 12 G
CDKD
Os05g32600 Orysa;CDKD1 R2 5 + 17909000 CB679198.1 7 G
CDKE
Os10g42950 Orysa;CDKE1 10 - 22379000 AU097604.1 16 G
CYCA
Os12g20320 Orysa;CYCA1;1 12 - 11705000 C28985.1 12 W,L,G
Os01g13240 Orysa;CYCA1;2 1 - 7377000 (C28985.1) 11 W,L,G
Os01g13260 Orysa;CYCA1;3 1 - 7389000 dbjAK063476 11 L
Os12g31810 Orysa;CYCA2;1 12 + 18670000 dbjAK106653 12 W,L,G
Os12g39210 Orysa;CYCA3;1 12 + 23522000 dbjAK110937 7 W,L,G
Os03g41100 Orysa;CYCA3;2 3 - 22157000 - 8 W,L,G
CYCB
Os02g55720 Orysa;CYCB1;1 2 + 24711000 (dbjAK107209) 11 W,L,G
Os01g17400 Orysa;CYCB1;2 1 - 10011000 dbjAK107209 7 W,L,G
Os05g41390 Orysa;CYCB1;3 5 + 23426000 CA762938.1 10 W,L,G
Os01g59120 Orysa;CYCB1;4 1 - 33936000 C25938.1 10 W,L,G
Os04g47580 Orysa;CYCB2;1 4 - 27200000 dbjAK070211 10 W,L,G
Os06g51110 Orysa;CYCB2;2 cycOs2 6 + 29606000 dbjAK070518 11 W,L,G
CYCD
Os08g32540 Orysa;CYCD1;1 8 - 19500000 dbjAK111449 6 W,L,G
Os06g12980 Orysa;CYCD1;2 6 + 7076000 dbjAK064081 6 L,G
Os09g21450 Orysa;CYCD1;3 9 + 11639000 CB679124.1 6 NI
Os09g02360 Orysa;CYCD3;1 9 + 489000 dbjAK103499 3 W,G
Os07g42860 Orysa;CYCD4;1 6 - 5976000 dbjAK103765 6 W,L,G
Os06g11410 Orysa;CYCD4;2 8 + 22888000 dbjAK070025 6 W,L,G
Os08g37390 Orysa;CYCD4;3 9 + 16137500 CB676959.1 6 L
Os09g29100 Orysa;CYCD4;4 3 - 15354000 - 9 L
Os03g27420 Orysa;CYCD4;5 7 - 25336000 dbjAK063671 6 L
Os03g10650 Orysa;CYCD5;1 3 - 5419000 CB644465.1 5 W,L,G
Os03g42070 Orysa;CYCD5;2 3 - 22705000 dbjAK068729 5 L,G
Os12g39830 Orysa;CYCD5;3 12 - 24000000 dbjAK101602 5 L,G
Os07g37010 Orysa;CYCD6;1 7 - 21865000 - 7 W,L,G
Os11g47950 Orysa;CYCD7;1 11 - 24768000 - 5 L,G
CYCH
Os03g52750 Orysa;CYCH1 3 + 28870000 dbjAK101854 13 W,L,G
CKS
Os03g05300 Orysa;CKS1 3 - 2554000 AU031496.1 3 G
RB
Os08g42600 Orysa;RB1 8 - 26080000 - 18 G
Os11g32900 Orysa;RB2 11 + 15792000 dbjAK064987 18 G
DEL
Os06g13670 Orysa;DEL1 6 - 7556000 CB656421.1 10 G

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Os02g50630 Orysa;DEL2 2 - 30147000 - 7 G
DP
Os10g30420 Orysa;DP1 10 - 15045000 - 9 G
Os03g05760 Orysa;DP2 3 + 2856000 CB664894.1 9 G
Os01g48700 Orysa;DP3 1 - 27711000 BI802607 9 G
E2F
Os02g33430 Orysa;E2F1 2 + 19775000 dbjAK068689 15 G
Os12g06200 Orysa;E2F2 12 + 2883000 dbjAK059233 14 G
Os04g02140 Orysa;E2F3 4 - 633000 - 13 G
Os04g33950 Orysa;E2F4 4 - 19665000 dbjAK067581 14 G
KRP
Os02g52480 Orysa;KRP1 2 + 31741000 AU172982.1 4 G
Os06g11050 Orysa;KRP2 6 - 5700000 - 4 G
Os11g40030 Orysa;KRP3 11 + 19889000 dbjAK106375 3 G
Os10g33310 Orysa;KRP4 10 - 16735000 dbjAK073804 3 G
Os03g04490 Orysa;KRP5 3 + 2072000 dbjAK064723 3 G
Os01g37740 Orysa;KRP6 1 - 22767000 AP003443.4 2 G
WEE
Os02g04240 Orysa;WEE1 2 - 1800000 - 10 G

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Table 2. Characteristic protein sequence signatures in CDK and cyclin genes in​ Oryza
sativa
Gene name Sequence signature Destruction box Other sequence
regions
CDKA
Orysa;CDKA1;1 PSTAIRE
Orysa;CDKA1;2 PSTAIRE
Orysa;CDKA2;1 PSTAIRE
CDKB
Orysa;CDKB1;1 PPTALRE
Orysa;CDKB1;2 PPTALRE
CDKC
Orysa;CDKC;1 PITAIRE
Orysa;CDKC;2 PITAIRE
CDKD
Orysa;CDKD1 NFTALRE
CDKE
Orysa;CDKE;1 SPTAIRE
Cyclin A
RVALSNISNV, no
Orysa;CYCA1;1 MRAILIDW, LVEVAEEY PEST
RVALVNITNV, no
Orysa;CYCA1;2 MRAILIDW, LVEVAEEY PEST
RVALSNISNV, no
Orysa;CYCA1;3 MRAILIDW, LVEVAEEY PEST KYEE
RTVLKDVTNI, no
Orysa;CYCA2;1 MRGILIDW, LVEVSEEY PEST KYEE
RVALSDLPTLSNA, no
Orysa;CYCA3;1 MRAILVDW, LVEVADEY PEST KYEE
RVALSELPTLSNN, no
Orysa;CYCA3;2 MRGILVDW, LVEVAEEY PEST KYEE
Cyclin B
KYEE,
Orysa;CYCB1;1 MRAILTDW, HYRL RRALADVSN, no PEST TSILTKCSRASG
KYEE,
Orysa;CYCB1;2 MRAILTDW, HYRL RRALADVSN, no PEST TSILTKCSRASD
KYEE,
Orysa;CYCB1;3 MRAILADW, HYKF RQALGDIGN, no PEST TSVLTARSKHAC
KYEE,
Orysa;CYCB1;4 MRAILADW, HHKF RRALGEIGN, no PEST TSVLTARSKVAC
Orysa;CYCB2;1 MRAILIDW, HYKL RRALRDIKN, PEST KYEE
Orysa;CYCB2;2 MRGILIDW, HYKL RRALRDIKN, no PEST KYEE
Cyclin D
LLCTE, ILKVRELY,
Orysa;CYCD1;1 RADSVAW no PEST KMEE
Orysa;CYCD1;2 LLCGE, ILKVRSVH PEST KMEE
LLCAE, ILKVQEYN,
Orysa;CYCD1;3 RAESVSW no PEST KMEE
LYCPE, ILTTRTRF,
Orysa;CYCD3;1 REAAVGW no PEST KVEE
LLCAE, IWKVHELY,
Orysa;CYCD4;1 RRDAIDW PEST KMEE

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 LLCAE, IWEVYTYY,
Orysa;CYCD4;2 RSEAIGW no PEST KMEE
LLCAE, IWKVHSYY,
Orysa;CYCD4;3 RMDAIDW no PEST KMEE
LLCEE, ICKVQAYY,
Orysa;CYCD4;4 RIAAIDW PEST KMEE
LLCAE, ICKVHSYY,
Orysa;CYCD4;5 RKDAIDW no PEST KMEE
LICRE, IVKGGGGA,
Orysa;CYCD5;1 RAWCVGW PEST KVEE
LMCQE, ILETRGCF,
Orysa;CYCD5;2 RATVKW no PEST KMEE
LTCQE, ILETRGYF,
Orysa;CYCD5;3 RLAAVKW no PEST KMEE
Orysa;CYCD6;1 ISKVRYDG, RREAAGF no PEST
Orysa;CYCD7;1 LYCDE PEST
Cyclin H
Orysa;CYCH1 MRVFYEQK

37
Table 3. Motifs found by the FootPrinter program in promoters of different​ Oryza sativa
core cell cycle genes
Gene name Motifs found by FootPrinter
CDKA
CTTCCTC (-81, -105), CTCCTCT (-86, -110), AAGCGAG (-226), ATAAAAG (-262),
Orysa;CDKA;1;1 CATAAAA (-263), AAGAGAG (-273), AAAAAAA (-351, -382)
TTTCTCC (-72), CCATTTC (-75), AAGAGAG (-220), AGAGAAG (-273), AAAAAAA
Orysa;CDKA;1;2 (-335, -382)
Orysa;CDKA;2;1 CTCCTCT (-62), CAAAAAT (-305)
CDKB
Orysa;CDKB1;1 TGAAAAA (-310)
AATTTGA (-217), TGAAAAA (-361), TTAAAAA (-368), AAATAAA (-378), TAAAAAA
Orysa;CDKB1;2 (-382)
CDKC
Orysa;CDKC;1
Orysa;CDKC;2
CDKD
Orysa;CDKD;1 AAAAAAAA (-285), GAAAAAAA (-288), AAACTTTG (-661), GTGATTTT (-860)
CDKE
Orysa;CDKE;1 TTTTTGTT (-531), TTTTTTGT (-532), ATTTTTTG (-533)
Cyclin A
Orysa;CYCA1;1
Orysa;CYCA1;2
Orysa;CYCA1;3
Orysa;CYCA2;1
Orysa;CYCA3;1
Orysa;CYCA3;2
Cyclin B
Orysa;CYCB1;1 TTTTCTCT (-408), TTTTTTTT (-783), ACTAAGAA (-836), GAAATTTG (-1140)
Orysa;CYCB1;2 ATTTTTTT (-674), ATTTTATT (-817), CATCTTCT (-1009)
Orysa;CYCB1;3 ATAATTTT (-759), TTAGTCAT (-806), GTTTTTTT (-1203)
Orysa;CYCB1;4 TTTATTTT (-765), TTTTATTT (-766), GATTTTAA (-1282)
Orysa;CYCB2;1
Orysa;CYCB2;2
Cyclin D
Orysa;CYCD1;1
Orysa;CYCD1;2
Orysa;CYCD1;3
Orysa;CYCD3;1 TCCTCTTC (-62), AATAAAAA (-179), CAATAAAA (-235), AAAAGACA (-362)
Orysa;CYCD4;1 TCTATTT (-673), TTTTTCT (-1011), TTATAAA (-1131), AAAACAA (-1146)
Orysa;CYCD4;2 AATTTTA (-903 -966)
Orysa;CYCD4;3 CCTCCTC (-121, -124), TTTTCAA (-570), TTTATAT (-1099)
Orysa;CYCD4;4 TATTTTA (-950)
Orysa;CYCD4;5 ATCAATA (-78), CTCACTC (-160), TCTATTT (-655)
Orysa;CYCD5;1 TAAATAA (-645), AAAAAAA (-654), ATTTATT (-739), ATTTAAA (-762)
Orysa;CYCD5;2 AAAATTA (-441), AAGATAA (-631), TTTTTTT (-1378)
Orysa;CYCD5;3 ATTCAAA (-375), ATTTAAA (-389), AATATAA (-700), TTGAAAT (-1505)
Orysa;CYCD6;1
Orysa;CYCD7;1 CTCCTTC (-198), TGCATGT (-436), TTGCATG (-466)
Cyclin H
Orysa;CYCH;1
CKS
Orysa;CKS;1 CGTTTCTT (-117), TTTTCCTC (-146), AAATTCAA (-478), AAAATTGA (-590)
RB
Orysa;RB;1 CAACCAAAC (-196), TTCTATTTT (-688), TGAGAATGT (-1601)
Orysa;RB;2
DEL
Orysa;DEL;1 AAGAAAA (-566), CTCCAAC (-1480), GTTGTGG (-1872)
AGATCCA (-98), AAATCAA (-225), TTTGAAA (-258), TCGACAA (-1046), TGTTTGG
Orysa;DEL;2 (-1941)
DP

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Orysa;DP;1 TTCAATAA (-766), ATTTTAAA (-817), GGAAGAGA (-1436)
TGTTATAG (-462), ATGAAAAG (-448), ACTTTTTA (-814), TAAAATCA (-853),
Orysa;DP;2 ATCAATCA (-1741)
Orysa;DP;3 TTTTTAAA (-699), TTTTTCAA (-848), TATCAAGT (-1188)
E2F
Orysa;E2F;1
Orysa;E2F;2
Orysa;E2F;3
Orysa;E2F;4
KRP
Orysa​;KRP1 CTTGTTG (-40), CTTAGCT (-107)
Orysa​;KRP2 ATTATTC (-115)
Orysa​;KRP3 TTTTTTT (-16), CGTCTTT (-133)
Orysa​;KRP4 TTTCTCT (-226), CATTTTG (-318)
Orysa​;KRP5 ATTATTC (-52), TTTCTCT (-168)
Orysa​;KRP6 AGGGAGA (-69), TTTATAT (-189), CATCTTT (-193)
WEE
CTTCCTC (-81, -105), CTCCTCT (-86, -110), AAGCGAG (-226), ATAAAAG (-262),
Orysa;WEE;1 CATAAAA (-263), AAGAGAG (-273), AAAAAAA (-351, -382)

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Table 4. Kinematic parameters determined for rice leaf growth and corresponding obtained
measurements.
Parameters Measurements
Final leaf length (cm) 45.7 ± 0.6
Leaf elongation duration (h) 118.2 ± 1.4
Leaf elongation arte (mm.h​-1​) 3.9 ± 0.05
Mature cell length (​μm​ ) 80.9 ± 0.67
Cell production rate (cell.h​-1​) 48.4 ± 1.85
Cell division rate (cell.cell​-1​.d​-1​) 1.03 ± 0.05
Cell cycle duration (h) 16.5 ± 0.67

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Table 5. List of genes whose expression levels are low modulated (less than 4 fold) in leaf

Constitutive or low fluctuating throughout the leaf

CDKA1;1 CYCD1;3 DP3 KRP4 CKS1
CDKA1;2 CYCD4;4 E2F3 KRP3
CDKC;1 CYCD7;1 E2F4
CDKC;2 CYCB1;2
CDKD;1 CYCH1
CDKE;1

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