JXB Advance Access published online on March 26, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm022
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RESEARCH PAPER |
Dicot and monocot plants differ in retinoblastoma-related protein subfamilies
Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, H-6726, Temesvári krt. 62, Hungary
* To whom correspondence should be addressed. E-mail: dudits{at}brc.hu
Received 24 August 2006; Revised 16 January 2007 Accepted 22 January 2007
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Abstract |
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The present study supports the view that the retinoblastoma functions are shared by two distinct retinoblastoma-related (RBR) protein subfamilies in the monocot cereal species, whereas dicot plants have only a single RBR protein. Genes encoding RBR proteins were identified and characterized in alfalfa (Medicago sativa), rice (Oryza sativa), and wheat (Triticum aestivum). The alfalfa MsRBR gene encodes a new member of the dicot RBR proteins (subfamily A). A comparison was made of two rice genes, OsRBR1 (subfamily B) and OsRBR2 (subfamily C), which exhibit differences in exon–intron organization and share only 52% amino acid sequence identity. The plant RBR proteins can be categorized into three distinct subfamilies, in which the similarity between members is greater than the similarity to other RBR proteins from the same species. Comparison of the transcript levels in various tissues revealed that the expression of the OsRBR1 gene was high in embryos or cultured cells and gradually decreased from the basal region to the tip of the leaves. The OsRBR2 gene displayed more transcripts in differentiated tissues, such as leaves and roots. In contrast, the mRNA level of the MsRBR gene did not differ significantly in either mature leaves or cultured cells. The results of yeast two-hybrid pairwise interaction assays demonstrated differences between the rice RBR variants in the interactions with the phosphatase 2A B'' regulatory subunit and an unknown protein. The in silico and functional data presented in this work highlight considerable differences between dicot and monocot species in the retinoblastoma regulatory pathways and permit an improved classification of RBR proteins in higher plants.
Key words: Alfalfa, NAC protein, protein phosphatase, retinoblastoma, rice, wheat
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Introduction |
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Recent discoveries in plant cell cycle research have supported the roles of both phylogenetically conserved and plant-specific regulatory mechanisms in the control of cell division. The identification of plant homologues of retinoblastoma-related (RBR) genes and the characterization of different components of the plant retinoblastoma (RB) pathway have demonstrated that the regulators of cell division can be targets for evolutionary changes that contribute to essential features in developmental strategies or environmental responses.
In mammalian cells, pocket proteins include the RB susceptibility gene product (pRB/p105) and the related proteins p107 and pRB2/p130 (Lee et al., 1987; Ewen et al., 1991; Mayol et al., 1993). These proteins have functions in the cell cycle regulatory steps that link cyclin–cyclin-dependent kinase (CDK)-integrated positive and negative growth signals to E2F-DP transcription factor activity in order to initiate or stop cell division (Xiao et al., 2003). Pocket proteins interact with a wide range of proteins involved in cellular differentiation and cell cycle control, and probably act as a scaffold to facilitate or bridge other protein–protein interactions (Weinberg, 1995; Dyson, 1998; Brehm, 1999). RB genes have been isolated from a variety of animal species: mouse (Bernards et al., 1989; Pertile et al., 1995; Huppi et al., 1996), Xenopus (Destree et al., 1992), chicken (Boehmelt et al., 1994), rat (Sawada et al., 1997), zebrafish (accession no. AAM91029), and fruitfly (accession nos NM_080297, NM_079648). Drosophila RBF proteins contribute to the cell cycle control in insects (accession no. AF197059) (Du et al., 1996).
The first plant RBR homologue in maize was identified by the characterization of a partial maize cDNA clone (Grafi et al., 1996). The RBR genes from Zea mays, ZmRBR2a and ZmRBR2b (Ach et al., 1997), are partial cDNA sequences which may represent alternative splice variants of the ZmRBR2 gene. The degree of homology between the proteins encoded by these ZmRBR genes is 
90%. The predicted proteins display homology to the pocket A and B domains of the animal RBR protein family (Grafi et al., 1996; Xie et al., 1996; Ach et al., 1997). Recognition of the RB-binding LxCxE motif in plant D-type cyclins (Dahl et al., 1995; Soni et al., 1995) or in the viral replication protein RepA (Xie et al., 1995) further strengthened the expectation that the RB pathway may also function in plants. A set of experiments demonstrated interactions between the maize ZmRBR1 and a variety of plant proteins such as D-type cyclins (Ach et al., 1997; Huntley et al., 1998), other cyclins (Roudier et al., 2000), viral proteins (Xie et al., 1996; Ach et al., 1997; Horváth et al., 1998), E2F transcription factors (Huntley and Murray, 1999; den Boer and Murray, 2000; Gutiérrez et al., 2002), and chromatin-associated proteins, e.g. histone deacetylases (Rossi and Varotto, 2002). Similarly, as in animals, RBR proteins can be phosphorylated by cyclin–CDK complexes in plants (Nakagami et al., 1999; Boniotti and Gutiérrez, 2001; Nakagami et al., 2002).
Only limited data are available on the mechanisms controlling the amounts of the RBR proteins and the expression pattern of the RBR genes in plant organs. The RBR protein has been shown to be synthesized both in proliferating and in differentiated tissues (Xie et al., 1996; Ach et al., 1997; Huntley and Murray, 1999). Developing maize leaves exhibit a gradient of proliferating and differentiated cells. The actively dividing cells are located at the leaf base, while the differentiated cells are formed toward the tip and the ZmRBR protein is detected mainly in the differentiated cells (Huntley et al., 1998).
After the discovery of the plant homologues of the RB gene in maize, an increasing number of plant genes encoding RBR proteins were isolated and characterized. Plant RBR cDNAs have been cloned from the dicot species Nicotiana tabacum (Nakagami et al., 1999), Chenopodium rubrum (Fountain et al., 1999), Arabidopsis thaliana (Kong et al., 2000), Pisum sativum (accession no. BAA88690), Euphorbia esula (accession no. AAF34803), Populus tremulaxPopulus tremuloides (accession no. AAT61377), and Scutellaria baicalensis (accession no. AB2051361), and from the monocot Cocos nucifera (accession no. AAM77469).
A striking degree of conservation exists in terms of the domain organization of the RB family members, including animal and plant species (Durfee et al., 2000). The conservation of RBR proteins over the A and B pocket domains raised the possibility that their function might also be conserved at the molecular level. Furthermore, the order of the conserved domains is similar in all RB proteins identified. The N-termini of RB proteins are highly variable in length and sequence, which may determine the specificity of these regulatory proteins.
Recent studies on plant cell division have discussed the significance of the presence of several RBR genes in maize (Sabelli et al., 2005; Sabelli and Larkins, 2006). In the present study, the comparative analysis of the RBR protein family was extended to monocots and dicots. The full-length cDNA sequence of a Medicago RBR gene and two RBR homologues in rice and wheat is reported. The data presented on the gene structure, the gene expression, and the comparison of amino acid sequences and interactors support the conclusion that two distinct plant RBR proteins function in monocot Gramineae species, while the dicots have only a single gene encoding the RBR protein, as is indicated by the complete genome sequences of Arabidopsis and Medicago.
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Materials and methods |
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Cloning of the Medicago sativa RBR gene (MsRBR)
To clone the MsRBR gene, a 305 bp PCR fragment was used which was obtained by amplification of a
ZAPII cDNA library using the following primer combination: forward primer 5'-TCCAAGTTGTACTATAGG-3', and reverse primer 5'-TGCCATGCTTTCCAAAAG-3'. The primers were designed on the basis of the conserved A domain part of the published ZmRBR2a sequence (Grafi et al., 1996). The ZAPII cDNA library was constructed from the mRNA of 3- to 4-d-old non-nitrogen-fixing nodules of Medicago sativa ssp. varia (Savouré et al., 1995), kindly provided by Dr Éva Kondorosi (Institut des Sciences Végétales, CNRS, Gif-sur-Yvette Cedex, France).
Cloning of two Oryza sativa RBR genes (OsRBRs)
For isolation of the rice RBR genes, a 1521 bp long MsRBR probe was used, which contained the conserved B domain and the C-terminal region of the MsRBR. A PCR-based cDNA library was prepared by using the SMARTTM cDNA Library Synthesis Kit (CLONTECH), and was kindly provided by Crop Design NV (Ghent, Belgium). The positive clones were selected and sequenced. The cDNA library screen resulted in retrieval of the OsRBR1 (accession no. AY941774) and OsRBR2 (accession no. AY941775) genes. Templates for sequencing were constructed by subcloning.
Identification of Triticum aestivum RBR genes (TaRBR1 and TaRBR2)
The rice OsRBR1 and OsRBR2 protein sequences were used in a TBLASTN search of the TIGR (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=wheat) wheat (Triticum aestivum) database. TCs (tentative consensus sequences) (TC52591, TC44330, TC39220, TC52174, and TC46259) with significant similarity were found. The remaining sequence of the TaRBR1 and TaRBR2 cDNAs was obtained with the PCR primers TaRBR1F (forward 5'-GAATTCGGCACGAGGCAGGCAGAGT-3'), TaRBR1R (reverse 5'-CAATTAAAACAAGGTGAAGTTTTACCCCACTGC-3'), TaRBR2F (forward 5'-GTTTTCATTCAAATTTTGAGAAGTT-3'), and TaRBR2R (reverse 5'-GCAGGATCGGAAGAGGCAGGGACAC-3'), after which the amplified fragments were sequenced.
Sequence alignment; phylogenetic analysis
TBLASTT analysis of the available genomic sequences was carried out to identify the exon–intron organization of the examined RBR genes, and the STRECHER program (http://bioweb.pasteur.fr/seqanal/interfaces/stretcher.html) was used to identify the borders. The regions containing RBR-like protein sequences from the A pocket domain up to the C-terminus were aligned by using ClustalW (http://bioweb.pasteur.fr/seqanal/ClustalW.php). The alignment was submitted to the PHYLIP server (http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html) (Felsenstein, 2002). The protpars feature was used with bootstrapping performed before analysis. The consensus tree was then displayed with bootstrap values.
RNA extraction and real-time RT-PCR
Leaves (10 cm long) from 2-week-old seedlings and 30 cm expanded leaves were collected from rice plants grown in the greenhouse. The tissues were frozen in liquid nitrogen and stored at –80 °C before RNA extraction. Total RNA was isolated by using the TRI Reagent method (Chomczynski and Sacchi, 1987) optimized for plant RNA extraction. RNA was quantified by means of spectrophotometric OD260 measurements, and the quality was assessed by calculating the ratio OD260/OD280 and by electrophoresis on a 1% formaldehyde agarose gel followed by ethidium bromide staining. RNA samples were stored at –80 °C.
Two-step real-time reverse transcription (RT)-PCR was performed. First-strand cDNA was synthesized from 1 µg of total RNA, using the M-MuLV H minus reverse transcriptase system (Fermentas, Vilnius, Lithuania) with random hexamer primers, according to the manufacturer's instructions. The cDNAs were diluted 1:3.5 with nuclease-free water. Aliquots of the same cDNA sample were used for all primer sets during real-time PCR, and amplification reactions were performed with all primer sets during the same PCR run. Gene-specific primers were designed for the sequences corresponding to the N-terminal regions of RB proteins. The specific primer pair (forward 5'CTCGTCAGGCTTAGATGTGCTAGAT-3', reverse 5'TGAACAATGCTGAGGGATTCAA-3') for the rice actin 1 gene (accession no. X63830) was used as internal control. In the case of alfalfa, the actin gene was also used as internal control. The specific primers for the examined genes were: MsRBR (forward 5'CCCAAGCCTTCCTGACATGT-3', reverse 5'TTGGATGACCGTAATGGAGAGA-3'); OsRBR1 (forward 5'CTCCTATCCAGCATGTCATCCTT-3', reverse 5'AGCCTTGACACACAGTAAAGAACAA-3'); and OsRBR2 (forward 5'TGGCCCCTCCGTGTGA-3', reverse 5' TGACGCCATGAAAGAGCTACA-3'). Each oligonucleotide hybridized specifically with cDNAs and produced correctly sized PCR fragments. Specific primers for actin cDNA were used as internal control for real-time PCR in all tested species. Reactions were carried out in a volume of 20 µl containing 150 nM of each primer, 7 µl of cDNA sample (derived from 40 ng of input total RNA). and 10 µl of 2x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) in a 96-well reaction plate, using the parameters recommended by the manufacturer (10 min at 95 °C and 45 cycles of 95 °C for 15 s and 60 °C for 1 min). Each PCR was performed in triplicate, and no-template controls were included. The specificity of the amplifications was verified at the end of the PCR run, using ABI Prism Dissociation Curve Analysis Software. The efficiencies of the amplicons were analysed by a standard curve method. Two independent experiments were performed, including reverse transcription and real-time PCR steps. The 2–
Ct method was used to analyse the real-time PCR data (Livak and Schmittgen, 2001). The expression of the examined genes was normalized to the endogenous control, in this case to the rice and Medicago actin genes, to compare the expression levels relative to a calibrator sample, i.e. the mature leaf tip; the Ct for the mature leaf tip was subtracted from the Ct for each sample. The expression level was also normalized with the aid of an absolute quantification method based on the standard curve, using a known amount of template DNA during real-time PCR detection (Rutledge and Cote, 2003). The relative expression values matched the expression values observed during use of the 2–Ct method.
Yeast two-hybrid matrix analysis
For yeast two-hybrid experiments, the vector constructs pGBT9/MsRBR, pBD-GAL4 2.1/OsRBR1, and pBD-GAL4 2.1/OsRBR2 were used as bait, and pAD-GAL4 2.1/MSV RepA (Horváth et al., 1998), pAD-GAL4 2.1/OsRBRI2 (unpublished results), pAD-GAL4 2.1/OsRBRI5 (unpublished results), pAD-GAL4 2.1/Medtr;CycD5 (unpublished results), and pAD-GAL4 2.1/MsPP2AregB (unpublished results) were used as prey. The cloning vectors pGBT9, pBD-GAL4 2.1, and pAD-GAL4 2.1 were obtained from Clontech and Stratagene (La Jolla, CA, USA). The Saccharomyces cerevisiae yeast strain PJ69-4a (James et al., 1996) was transformed with the appropriate constructs, as performed by Schiestl and Gietz (1989). The transformants were grown on a –Trp, –Leu, –His, –Ade medium, with monitoring of the activation of the HIS3 and ADE2 reporter genes. The protein association strength was quantified with the ONPG (o-nitrophenyl-D-galactopyranoside) method. Relative ß-galactosidase units were determined according to Horváth et al. (1998).
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Results |
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Identification of RBR genes in alfalfa, rice, and wheat
Various primer combinations were designed and tested for PCR amplification of the RBR gene from M. sativa, on the basis of the published sequence information on the maize RBR gene. The PCR product was sequenced and used for the screening of a cDNA library. Following this approach, a full-length cDNA MsRBR was obtained which was 3559 bp long; and the encoded protein consisted of 1025 amino acids, with a calculated molecular mass of 114 kDa and a pI of 7.6 (Table 1). Alignment of the predicted amino acid sequence of MsRBR with several members of the RBR protein family revealed reliable homology, especially in the A and B pocket domains (Fig. 1). MsRBR shares 67–91% amino acid sequence identity with full-length dicot RBR sequences.
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Two rice RBR genes were also isolated via a rice cDNA library screen, using the coding region of the Medicago RBR gene. Analysis of the full 3498 bp OsRBR1 sequence demonstrated that this cDNA encodes a protein of 1010 amino acids with a molecular weight of 111.5 kDa and a pI of 8.65. OsRBR1 shares 52–85% identity at the amino acid level with full-length plant RBR proteins. By further cDNA library screening, another rice RBR variant, OsRBR2, was identified. The nucleotide sequence of the OsRBR2 gene displayed only 52% identity to OsRBR1. The full-length OsRBR2 cDNA clone was 3383 bp long and encoded a polypeptide of 978 amino acids with a calculated molecular weight of 107.9 kDa and a pI of 8.10. The OsRBR1 and OsRBR2 proteins share 52% identity in amino acid sequence, which indicates the presence of two distinct RBR proteins and coding genes in rice that belong in different classes of RBR proteins characteristic for the monocot species, as shown in Figs 1 and 3.
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Database homology searches were used to identify other monocot orthologous counterparts. The known sequence data on the RBR proteins from rice were used as queries to perform TBLASTN searches against the available wheat expressed sequence tag (EST) database. The partial sequence information on the identified TCs helped in the isolation of RBR genes from wheat by PCR amplification and sequencing of cDNAs. Analysis of the 3446 bp sequence from TaRBR1 indicated that the full-length sequence encodes a protein of 957 amino acids with a calculated molecular mass of 106.1 kDa, while the identified partial TaRBR2 cDNA was 1890 bp and the partial encoded protein contained 598 amino acids (Table 1). The wheat RBR proteins share identical amino acids to an extent of 58%. This low level of homology reflects the presence of two distinct classes of RBR genes in this Gramineae species. Specific motifs of conserved regions can be recognized in the protein alignment (Fig. 1).
Exon–intron organization of plant RBR genes
The availability of the A. thaliana and rice genome sequences allowed comparisons of the structures of selected dicot and monocot RBR genes. The RBR gene in Arabidopsis was identified by searching the TAIR database (Kong et al., 2000). The chromosomal location of each RBR gene was determined by using different databases: the rice physical map (Chen et al., 2002), the genomic sequences of Arabidopsis (http://www.arabidopsis.org), and the available Medicago BAC (bacterial artificial chromosome) sequences (http://www.genome.ou.edu/MtBACs.html). It was established that OsRBR1 mapped to chromosome 8 (BAC clone: Osch8 P0666G10), and that OsRBR2 was located on chromosome 11 (BAC clone: Osch11 OsJNBa0080108). Figure 2 shows the exon–intron boundaries and the location of the A and B pocket domains in Arabidopsis (AtRBR), Medicago (MsRBR), and rice OsRBR1 and OsRBR2 genes. Three of the analysed RBR genes contain 18 exons in the approximately 8000 bp long DNA fragments. In the case of MsRBR, one intron is missing, and exon 14 is therefore as long as AtRBR exons 14 and 15. The presence of conserved RB domains is common to all plant RBR genes. The analysis of the positions of sequence domains revealed that the highly conserved coding region of the A pocket domain was located on exon 10 for all plant RB-like genes, the following exon (11) encoding the spacer region. In all examined plant RBR genes, the B pocket domains are coded by the next four exons. The intron between exons 10 and 11 in the rice OsRBR2 gene was longer than in other RBR genes. The OsRBR1 gene exhibits considerable similarity in exon–intron structures to the AtRBR gene; and their introns are located at similar positions within the coding regions (Fig. 2).
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Comparative phylogenetic analysis of the plant RBR proteins
To investigate the relationships between known members of the RBR protein family from different plant species, phylogenetic analyses were carried out on deduced protein sequences. Table 1 lists previously characterized plant RBR proteins, with the name, the number of amino acid residues, the molecular mass, and the pI value. The published partial RBR protein sequences from maize (ZmRBR2a and ZmRBR2b) can represent alternatively spliced variants of ZmRBR2 (Grafi et al., 1996). Furthermore, ZmRBR1 and ZmRBR2 are differentially spliced transcripts, the homology between these ZmRBR proteins being higher than 90%.
The ClustalW method was used for alignment to classify various representatives of RBR proteins from diverse plant species. As the phylogenetic tree created by the PHYLIP method shows, the plant RBR proteins can be categorized into three distinct subfamilies (Fig. 3). Subfamily A contains all dicot RBR proteins, such as AtRBR, EeRBR CrRBR, MsRBR, NtRBR, PtRBR, and PsRBR, while subfamilies B and C comprise different monocot RBR proteins. The phylogenetic tree definitely separates members of subfamily B, including the OsRBR1/TaRBR1/ZmRBR3/CnRBR proteins, from subfamily C, including the OsRBR2/TaRBR2/ZmRBR1/ZmRBR2 proteins. The variants of the monocot RBR proteins in subfamily C were identified as a distinct cluster that had no counterpart in dicot species. The RBR from the non-graminaceous monocot C. nucifera, CnRBR (accession no. AAM77469), belongs in the monocot subfamily B and lies near the node of the branching point between dicots and monocots (Fig. 3). The dicot RBR proteins share 66–91% amino acid identity within subfamily A. The monocot RBR protein sequences are 69–85% identical in subfamily B and 74–90% identical in subfamily C. The orthologous proteins of the different cereal species generally exhibit greater similarity to each other within the subfamilies than to another RBR protein from the same species. Subfamilies A and B share 54–67% identity, while the sequences of the RBR proteins in subfamily C are 48–60% identical to those in the dicot subfamily A. The two distinct monocot subfamilies B and C share 48–59% amino acid identity. The present description of the subfamilies of plant RBR proteins reveals a phylogenetic separation between the known monocot RBR proteins, indicating a more complex RB pathway in the monocot cereal species. The phylogenetic tree presented in Fig. 3 suggest a closer relationship between the dicot RBR proteins (subfamily A) and monocot subfamily B proteins than between the subfamily C RBR proteins and the dicot representatives. The similarities and differences between the RBR proteins of various subfamilies will be discussed later.
Differential expression of plant RBR genes
The expression patterns of the different RBR genes in rice and alfalfa were investigated by real-time quantitative RT-PCR analysis of RNA samples from different tissues. In view of the high similarity between the homologous rice sequences, the primers were designed for the less homologous 5' regions, allowing specific amplification of the target genes.
Growing young rice leaves measuring 10±1 cm were cut into four sections, and six regions were cut from expanded rice leaves measuring 30±3 cm. In the case of alfalfa, three regions were cut from mature leaves. Figure 4 presents the relative mRNA amounts normalized to the actin RNA levels. Figure 4A shows the expression levels of the two rice RBR genes, which differ significantly in the various organs. The OsRBR2 transcripts were found to be more abundant in both the younger developing and the fully expanded leaves. The ratio between the transcripts of the OsRBR1 and OsRBR2 genes was dependent on the age of the leaves. In the young leaves, the constitutively expressed OsRBR2 gene was transcribed at a 5–10 times higher level than the OsRBR1 gene, while in the fully expanded leaves the OsRBR2 transcripts were present in quantities several hundred times higher than the mRNA levels of OsRBR1. In contrast to the OsRBR2 gene, the expression of the OsRBR1 gene decreased from the basal region to the tip region of the analysed leaves. This pattern suggests that the OsRBR1 gene is more active in the basal region of the leaves, where cell divisions are expected to occur and cell differentiation is not complete. In accordance with these findings, higher transcript levels of this gene were detected in embryos and actively dividing cultured cells. Analyses of the transcript accumulation in the various tissues of alfalfa demonstrated that this gene (subfamily A) was constitutively active in various sections of the mature leaves, and also in the cells of the roots and suspension cultures (Fig. 4B). The presented expression data reveal essential differences between the two rice RBR gene variants expressed differentially in various tissues, with variable ratios between dividing and differentiated cells.
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Selectivity of RBR proteins in interactions with functional protein partners
For yeast two-hybrid interaction studies, plasmids containing the full-length sequences of MsRBR (pGBT9/MsRBR), OsRBR1 (pBD-GAL4 2.1/OsRBR1), and OsRBR2 (pBD-GAL4 2.1/OsRBR2) were used as bait, and yeast strain PJ69-4A (James et al., 1996) co-transformed with vectors carrying the rice RBR interactor partners (OsRBRI2 and OsRBRI5), Medicago cyclin D5 (Medtr; CycD5), Medicago protein phosphatase PP2A B'' regulatory subunit, and maize streak virus RepA (MSV RepA) (Horváth et al., 1998) were used as prey. As shown in Fig. 5, all three plant RBR proteins interacted with the viral replication protein which contains the well-known LxCxE RB-binding motif. None of them was able to bind to the analysed Medicago D-type cyclin (Medtr; CycD5), which lacks this motif (data not shown). Extensive yeast two-hybrid screens, using the full-length MsRBR protein as bait, resulted in several Medicago truncatula interactors (encoded by sequences TC109726, BI270584 [GenBank] , and AW692851 [GenBank] ). One of these proteins exhibited homology to the PP2A B'' regulatory subunit (39% identity and 57% similarity to murine PR59 PP2A regulatory subunit) reported by Voorhoeve et al. (1999). Further pairwise assays revealed that this protein discriminated between the two rice RBR proteins in interaction strength. The alfalfa RBR and OsRBR1 proteins underwent strong association with the PP2A B'' regulatory subunit, while OsRBR2 failed to interact with this prey (Fig. 5). Further yeast two-hybrid screens performed with OsRBR1 as bait resulted in a new interacting protein, OsRBRI2, which exhibited significantly different association strengths with the analysed dicot and monocot RBRs. In this test, selective interactions were detected between OsRBRI2 protein and the MsRBR and OsRBR1 proteins. The specificity of this interactor toward only one of the rice RBR proteins may be an indication of functional differences between the rice RBR variants. On the basis of sequence database searches, OsRBRI2 is an expressed protein homologous to AK101154 [GenBank] (cDNA_clone_J033027D16). Another interacting partner, the OsRBRI5 protein, displayed homology to NAC proteins (LOC_Os02g38130, by TIGR Rice Genome Database). This interactor contains the RB-binding motif (LxCxE), and can therefore interact with all the analysed RBR proteins, but differences in the strengths of the interaction were detected. The similarity observed between the alfalfa MsRBR and the rice OsRBR1 as concerns the preference of the interactors can be interpreted as a sign of a closer functional relationship between RBR subfamilies A and B. The presented protein–protein interaction data support the suggestion that the functional diversity of the RBR proteins is characteristic in monocot rice.
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Discussion |
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Since the RBR proteins are expected to play significant roles in the control of plant growth and development, in recent years increasing attention has been paid to the RBR protein family of plants as molecular regulators (Durfee et al., 2000; Gutiérrez et al., 2002; Sabelli and Larkins, 2006). The present study is focused on the identification and initial characterization of new members of the RBR family in plants. Five cDNAs were isolated and designated MsRBR from the model legume M. sativa, OsRBR1 and OsRBR2 from rice, and TaRBR1 and TaRBR2 from wheat. The pocket proteins encoded by these genes belong in the RBR protein family whose members all share common A/B domains conserved in both animals and plants (Fig. 1).
Gene comparisons facilitate studies on the conservation of coding and non-coding regions and on the molecular mechanisms of genome evolution. In the human RB family, the RB transcripts are encoded by 27 exons, the p107 gene is composed of 22 exons, and the RB2/p130 gene consists of 22 exons (Claudio et al., 2002). It is evident that the p107 and RB2/p130 genes exhibit homology in their genomic arrangement and conserved protein regions, suggesting a closer evolutionary relationship between the p107 and RB2 genes. As in the human RB family, the exon–intron arrangements of plant RBR genes follow the observed protein similarities between the RBR proteins. The examined plant RBR genes contain 18 exons and 17 introns, and the homologous exons are identical in size and share high sequence identity at the nucleotide and amino acid levels. The transcribed region of the plant RBR genes is organized into 18 exons, and spans about 7000 bp of genomic DNA from the transcription start to the termination site (Fig. 2). The highly conserved coding region of the A pocket domain is located on exon 10 in all plant RB-like genes. Overall, the size and arrangement of the exons encoding conserved domains appear to be typical features of the RBR genes, as in all members of the vertebrate RB family. Analysis of the exon–intron structure indicates that the monocot RBR subfamily B is more closely related to the dicot genes (subfamily A), as shown by the phylogenetic tree (Fig. 3).
The constructed phylogenetic tree presented here, which consists of RBR proteins from 16 plant species, reveals that the plant RBR proteins can be classified into three subfamilies on the basis of the amino acid sequence similarity. Sequence analysis allows the separation of two characteristic subfamilies (subfamilies B and C) of RBR proteins from monocot species in addition to the cluster of dicot RBR proteins (subfamily A), as shown in Fig. 3. The organization of the MsRBR gene and the amino acid sequence of the encoded protein reveal that the alfalfa RBR protein belongs in subfamily A, which includes all known dicot RBR proteins deposited in the database to date (Fig. 3). Previously, A. thaliana was the only higher plant among these species known to have a RBR gene since the Arabidopsis genome had been completely sequenced (Seki et al., 2002). Extensive in silico studies on EST/TC databases support the suggestion that, like Arabidopsis, other dicot species contain a single RBR gene. This assumption was recently verified by an analysis of the complete genome sequence of M. truncatula (Cannon et al., 2006). The present search identified a single RBR gene on chromosome 8 (accession no. AC119410) of Medicago. Within the A and B pocket domains, the RBR proteins exhibit very high sequence identity, which is indicative of a common ancestor. In non-vascular plants, homologues to RBR genes have been identified, such as the mat3 gene (AF375824 [GenBank] ) from the unicellular green alga Chlamydomonas reinhardtii (Umen and Goodenough, 2001), the recently cloned RB-like (mat3) gene from Chlamydomonas incerta (AY781411 [GenBank] ), a putative RB homologue (AJ428952 [GenBank] ) from moss (Physcomitrella patens) (http://moss.nibb.ac.jp), and the newly reported RBR gene in Ostreococcus tauri (Robbens et al., 2005). All these studies also suggest that RBR genes have an ancient origin. The recognition of two cereal RBR proteins presumes gene duplication, which is prevalent in plants and which might lead to evolutionary innovation. The present comparative study provides a new example of the divergence of species belonging in the two major angiosperm subclasses, monocots and dicots.
In the animal kingdom, the complexity of the RB pathway is illustrated by the increasing number and functional diversification of the RBR proteins from insects to mammals. In insects such as Drosophila melanogaster, two RB-like proteins (Rbf1 and Rbf2) have been discovered and shown to have a distinct role in development (Keller et al., 2005). These gene products are co-expressed at several stages of development and their transcriptional induction has been linked to the mitotic programme in the eye imaginal disc. The Rbf2 protein level demonstrated temporal variation during embryogenesis, in contrast to the more constant levels of Rbf1 (Stevaux et al., 2002; Keller et al., 2005). The human RB family proteins represented by three orthologues, pRB/p105, p107, and pRB2/p130, which control the major G1 checkpoint, promote terminal differentiation or mediate regulation of the chromatin structure (Rossi et al., 2003), or repress the transcription (Zhang and Dean, 2001; Ho and Dowdy, 2002). The genes encoding the three RB family members vary in their expression patterns in different tissues. pRb2/p130 displays high mRNA levels in non-proliferating cells, while p107 accumulates in cells from the middle of G1 to mitosis. In plants, a diversification of the RBR genes and encoded proteins can be seen only in cereal monocot species.
The present study revealed the most characteristic differences between the two rice RBR genes in expression patterns detected in the leaves in various developmental stages. The transcripts of the OsRBR2 gene were detected in all the leaf tissues analysed, indicating the constitutive expression of this gene variant. Similarly, relatively high amounts of OsRBR2 mRNAs were accumulated in the root tissues. The transcript level of the OsRBR2 gene was significantly higher than the mRNA level of the OsRBR1 gene both in the segments of young leaves and in older rice leaves. These experimental data suggest a role for this gene variant in terminally differentiated cells. Despite the fact that leaf tissues consist of a variety of cell types, most of the cells are differentiated ones, and in root explants a majority of the cells are from the elongation zone and are expected to be non-cycling. The transcript of the other rice gene, OsRBR1, exhibits a different, but characteristic distribution pattern. There is a gradual reduction of the level of OsRBR1 mRNA from the basal section of the leaves toward the leaf tip, especially in older leaves. In general, 10 times more OsRBR1 mRNA is accumulated in the younger leaves. The difference is even more significant in the basal section of the leaves. As a consequence of their mode of development, monocotyledonous maize leaves display a proliferative gradient from the meristematic region at the leaf basal region through a series of progressively differentiating cells to the mature zone at the tip, made up largely of fully differentiated cells (Granier et al., 2000). It is presumed that rice leaves show a similar pattern of differentiation (Barrôco et al., 2006). This was also indicated by the previous observation that the monocot leaf is polarized in both cell division and expansion. In monocots, a basal meristem generates files of cells that remain aligned from the base to the tip of the leaf (Nelson and Langdale, 1989). As concerns the developmental pattern of cereal leaves and the expression profile of the OsRBR1 gene, a link is suggested between the activity of this gene variant and the potential of the cells to undergo division. It was recently demonstrated that NbRBR suppression by VIGS exerts effects on various cell layers at different rates (Park et al., 2005). The tissue- and cell-specific expression of pocket proteins has also been demonstrated in animals, and the three members of the RB family in humans were found to differ in their expression patterns (Claudio et al., 2002). The present gene expression data are in harmony with the suggestion that the different transcript profiles of the two rice RBR genes may reflect functional divergences, as in animals.
Studies on cell division in maize leaves have led to the finding that the rate of cell division is consistently high in the basal region of the developing leaves (Granier et al., 2000). This part of cereal leaves can be utilized for callus induction and plant regeneration through the reactivation of cell division by treatment with auxin (2,4-dichlorophenoxyacetic acid) (Zamora and Scott, 1983). In developing embryos and cultured cells, the OsRBR1 transcripts have been found to be more abundant than those from the counterpart OsRBR2 gene. Since these tissues are rich in dividing cells, these data lend support to the conclusion that the activity of the OsRBR1 gene may be related to cell division or cell cycle progression. In the mouse, the differential expression of RB and homologue genes has also been demonstrated during embryogenesis (Jiang et al., 1997). The accumulation of alfalfa gene MsRBR transcripts did not vary significantly in the different segments of the leaves (Fig. 4B). Similar mRNA levels were detected in the leaves and in the rapidly dividing cells in the suspension cultures, suggesting that the activity of this dicot RBR gene is independent of the actual division events occurring in the analysed tissues. The transcript analysis of the Arabidopsis RBR gene involving use of the Genevestigator database (Zimmermann et al., 2004) (www.genevestigator.ethz.ch) indicated that the AtRBR is expressed constitutively in a variety of Arabidopsis organs. A recent report on microarray analysis, focusing on the cell cycle genes in Arabidopsis leaf development, also showed the constitutive expression of the AtRBR gene when cells progress from proliferation to maturity (Beemster et al., 2005). The presented gene expression data emphasize the divergence of the rice RBR genes, which is a novel feature of cereal monocot species as compared with the dicot species exhibiting a single RBR gene, as in the case of alfalfa.
During evolution, proteins have evolved specifically to interact with different sets of partners. Selectivity in interactions can reflect the functional divergence of the RBR proteins from various subfamilies. D-type cyclins containing the LxCxE amino acid motif from a variety of plant species have been found to interact with RBR proteins (Huntley et al., 1998; Boniotti and Gutiérrez, 2001). In alfalfa, an A2-type cyclin interacted with the maize ZmRBR1 protein in a yeast two-hybrid system (Roudier et al., 2000). The interference of viral proteins with events in the cell cycle of the host cells can be achieved through their interactions with RBR proteins (Gutiérrez, 2000). The present yeast two-hybrid data (Fig. 5) indicate both similarity and diversity in the interactions between the RBR proteins from different subfamilies and various interactors. The RBR proteins from either subfamily can associate with the LxCxE motif-containing partners. One of these interactors is the OsRBRI5 protein, which belongs in the plant-specific NAC family (for NAM-ATAF-CUC-related). NAC family genes have been reported to be involved in various functions, such as organ development and morphogenesis, responses to virus attack and wounding, or signal transduction, such as auxin and abscisic acid signalling (Olsen et al., 2005). Further experiments are needed to evaluate the functional consequences of the RBR protein–NAC transcription factor interaction; however, it should be noted, that among Arabidopsis and rice NAC proteins, only the latter contains the RB-binding protein motif (LxCxE).
The regulatory functions of RB proteins in cellular processes depend essentially on their phosphorylation status, which is also regulated by phosphatases PP1 and PP2A (Yan and Mumby, 1999). The present yeast two-hybrid analysis has revealed that the MtPP2A B'' regulatory subunit, as a strong interactor of MsRBR, can also interact with the OsRBR1 protein, but there was no detectable association with the OsRBR2 protein. A similar situation was reported for mammalian pocket proteins. Murine RBs were shown to interact with the PR59 PP2A B'' regulatory subunit in a differential manner: only the p107 protein participated in a strong association; the pRb protein failed to form a complex (Voorhoeve et al., 1999). The PR59-associated PP2A complex exhibited dephosphorylation activity on bound p107 in vivo, and overexpression of this phosphatase regulatory subunit in U2-OS cells resulted in inhibition of the cell cycle progression and the arrest of cells in G1 phase (Voorhoeve et al., 1999). As regards the selectivity in complex formation, additional support is furnished by the fact that plant RBRs belonging in subfamilies B and C have different roles in the regulation of plant cell division and differentiation. This is in accord with the organization of the phylogenetic tree (Fig. 3), indicating a closer relationship in structure and interactions between the RBR proteins of subfamilies A and B. However, the RBR gene expression profiles differ in these subfamilies. The developmental regulation of RBR gene expression is limited only to the OsRBR1 gene (subfamily B), while the MsRBR gene (subfamily A) and the OsRBR2 gene (subfamily C) share similarity in constitutive expression profiles. Further studies are needed to establish evolutionary links between the RBR proteins of different subfamilies.
The identification of two types of monocot cereal RBR proteins with distinct features in gene organization, expression pattern, amino acid sequence identity, and interactor partners points to significant differences between dicot and monocot species as concerns the functional role of the RB pathway. The present report on the cloning of RBR genes and comparison of the encoded proteins from alfalfa, rice, and wheat extends our knowledge by classifying the RBR proteins into three subfamilies. Diversification of the RB functions in monocot cereals strengthens the view of a complex RB regulatory pathway in Gramineae. Further genetic, biochemical, and physiological studies are required for a functional characterization of the different members of the RBR protein family in plants and for a specification of the roles of various monocot RBR proteins.
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Acknowledgements |
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We thank Katalin Török for technical assistance and Mátyás Cserháti for his critical reading of the manuscript. G.V.H. is grateful for the support of a János Bólyai Research Fellowship. The authors are grateful to Dr David Durham for the improvement of the English text. This work was supported by the Crop Design NV (Ghent, Belgium).
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Abbreviations |
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RBR, retinoblastoma-related protein; TC, tentative consensus.
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