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Cloning and molecular characterisation of the maize retinoblastoma gene(ZmRBR2)

Manuela Najera-Martınez a, Elena Ramirez-Parra b, Jorge Vazquez-Ramos a, Crisanto Gutierrez b,Javier Plasencia a,*a Departamento de Bioquımica, Facultad de Quımica, Universidad Nacional Autonoma de Mexico, 04510 Mexico, D.F., Mexicob Centro de Biologıa Molecular ‘‘Severo Ochoa’’, Consejo Superior de Investigaciones Cientıficas, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain

1. Introduction

During the cell cycle, DNA is replicated to segregate identicalsets of chromosomes into the daughter cells. A key regulatory pointin the cell cycle is the G1–S transition at which the cell integratesexogenous and endogenous signals to proliferate, or differentiate.The retinoblastoma (RB)–E2F pathway plays a crucial role incontrolling G1–S transition, through a mechanism involving theinteraction of the retinoblastoma protein with the transcriptionfactor E2F, and by the recruitment of histone deacetylases toremodel chromatin and hinder transcription [1]. High conservationin this mechanism between mammals and plants has been inferredfrom the cloning and characterisation of the respective genes indistinct plant species [2,3]. The sequenced genome from the modelplant Arabidopsis thaliana predicts 12 cyclin-dependent kinase(CDK) genes, more than 30 different cyclin genes and 8 E2F/DPfamily genes [4], but only one retinoblastoma-related (RBR) gene[5]. Like in mammals, the retinoblastoma protein is phosphory-lated by a PSTAIRE CDKA/CYCD complex in a cell-cycle-dependent

manner [6] and its role in plant cell cycle control and developmenthas been demonstrated through various strategies. Silencing of theNicotiana benthamiana RBR results in plant growth retardation,abnormal organ development, prolonged cell proliferation [7] andeven cell death in mature tissues [8]. RBR protein titration by theinducible expression of the geminiviral RepA protein, showed thatat early stages of A. thaliana leaf development, RBR restricts celldivision and at later stages controls the endocycle [9]. RBR genesilencing by RNA interference in A. thaliana generates a root apexwith several extra cell layers developed in the columella root cap.These cells are undifferentiated and do not develop amyloplasts asdo mature columella cells, suggesting a role for RBR in maintainingstem cell identity as in mammalian cells [10]. A. thaliana mutantswith T-DNA insertions in the RBR gene results in high rates ofaborted ovules, supporting an important role for RBR in femalegametophyte development [11]. RBR cDNAs have been isolated inother dicot plants, such as N. tabacum [12], Chenopodium rubrum

[13] and Medicago sativa [14] and is a general consensus that asingle RBR gene is present in the dicotyledonous genomes [15].

In contrast, the situation in monocots is more complex than indicots, as maize, wheat and rice genomes contain at least twodistinct genes [14]. The first plant RBR (RBR1) gene was originallyisolated from maize [16,17] as a partial cDNA coding for a 75-kDa

Plant Science 175 (2008) 685–693

A R T I C L E I N F O

Article history:

Received 18 January 2008

Received in revised form 27 June 2008

Accepted 15 July 2008

Available online 30 July 2008

Keywords:

Zea mays

Gene expression

Intron

Cell cycle

Promoter

A B S T R A C T

Retinoblastoma-related (RBR) proteins have been described in plant cells, and have been shown to be

important in controlling plant growth, gametophyte and leaf development, and maintenance of stem cell

identity. Whereas all dicotyledonous plants studied so far contain a single RBR gene, monocotyledonous

species contain at least two genes and maize is the only known plant species with three RBR genes in its

genome suggesting a recent gene duplication event. We report the genomic structure of the ZmRBR2 gene

and its expression pattern. The ZmRBR2 transcript contained a 283-bp 50-UTR and a putative canonical

TATA box. The gene had a low GC content and showed conserved and unique features with other plant

RBR genes. ZmRBR2 gene promoter-GUS fusions, introduced in Arabidopsis thaliana plants, drove high GUS

activity in cotyledons, the shoot apical meristem and in flowering tissue, especially in anthers and pollen.

ZmRBR2 transcript levels were consistently 50% lower in several tissues than those of ZmRBR1, suggesting

a differential regulation of these two genes. Our results are consistent with the ubiquitous tissue

distribution and function of plant RBR proteins and emphasize differences between the monocot and

dicot RBR pathways.

� 2008 Elsevier Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: +52 55 5622 5275; fax: +52 55 5622 5329.

E-mail address: javierp@servidor.unam.mx (J. Plasencia).

Contents lists available at ScienceDirect

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0168-9452/$ – see front matter � 2008 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.plantsci.2008.07.004

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protein containing the A and B pocket domains that is capable ofinteracting with the LXCXE motif contained in the geminiviral RepA protein. Later on, the full-length ZmRBR1 cDNA and two partialcDNA clones (ZmRBR2), which have high identity (90%) in thecoding region with ZmRBR1, were cloned [18]. This protein islocalised in the nucleus and is capable of interacting with a plantcyclin D. More recently, a third distinct RBR cDNA (ZmRBR3) wasisolated from maize and may constitute a different type of RBR as itonly has 43% identity with ZmRBR1 and ZmRBR2 [19]. The RBR

gene is expressed in all tissues tested so far, including matureleaves, shoot apex, root, immature ear, silks, pollen, embryo, andendosperm, although transcript levels vary. The presence of threeRBR genes in maize suggests that differential expression of distinctmembers is temporally or spatially regulated during plantdevelopment [20]. Cloning of a full-length ZmRBR2 cDNA hasbeen a complicated task as other groups [18] and we haveattempted it unsuccessfully. As maize is the only plant speciesknown so far to contain three RBR genes, in this study we report thegenomic structure of the Zea mays retinoblastoma-related geneZmRBR2, thus completing the set of RBR genes in its genome. Thegene showed conserved and unique exon–intron arrangementpatterns as compared with other plant RBR genes and the predictedprotein had high identity with the previously described ZmRBR1

suggesting a recent gene duplication. However, ZmRBR2 transcriptlevels were found to be about half of those from ZmRBR1.

2. Materials and methods

2.1. Isolation of a maize Rb genomic clone

Approximately 5 � 105 plaque-forming units (pfu) from alambda Fix II genomic library (etiolated maize seedlings,Stratagene, La Jolla, CA), were screened with a 384 bp cDNA,containing the 50-end of the ZmRB1 cDNA [16] according toSambrook et al. [21]. Strong-hybridising plaques were excised torepeat the screening at several dilutions until clones that yielded100% of hybridising pfu signals were obtained. Each isolated pfuwas propagated and the DNA was extracted using a Qiagen-Lambda kit (Qiagen, Valencia, CA). The full-length insert wasreleased by digesting the purified DNA with NotI, yielding a 14-kbDNA fragment that was submitted to restriction analysis. An SstI9.4 kb fragment was cloned into SstI-digested pBluescript, produ-cing plasmid RB9000S that was propagated in Escherichia coli

DH5a strain. Purified plasmid DNA was obtained and used todetermine the nucleotide sequence by automatic DNA sequencing.

2.2. Primer extension analysis

Transcription initiation site of ZmRBR gene was mapped byprimer extension of leaf and stem RNA primed with 50-end labelledoligonucleotide P5: 50-ACTAATCCATGGGATGCAGG-30 (Fig. 1C) and200 units of Superscript-II for 1 h at 42 8C. The reaction productswere resuspended in loading buffer and resolved in a 4.5%polyacrylamide sequencing gel (45% urea) prepared in GTE (0.57 MTaurine, 10 mM Na2EDTA�2H2O, 1.78 M Tris-base) along with aDNA sequencing reaction as size marker. The gel was dried andexposed to X-ray film.

2.3. Genomic DNA blot analysis

Genomic DNA extracted from dry maize embryo axes [22] wasdigested (40 mg DNA per lane) with restriction enzymes BamHI,EcoRI, HindIII and SstI, and separated on a 0.8% agarose gel. DNAwas transferred onto nylon membranes and cross-linked with UVlight [21]. The membrane was hybridised with a 32P-labelled DNA

probe (clone RB1500) at 68 8C for 18 h in 6� SSC and washed athigh stringency before exposure to X-ray film.

2.4. Transgenic plants

Promoter activity was assayed by generating transgenic A.

thaliana (Columbia [Col-0] ecotype) plants expressing a ZmRBR2

promoter-b-glucuronidase (GUS) fusion. A DNA fragment spanningfrom positions�1372 to +311 (+1 is the transcription initiation site)was amplified from RB9000S clone using primers F71 (50-AAGC-TTACCGAGGTTCGGTTCTTG-30) and R81.5 (50-CCCGGGGGAGGG-TCCTGCGAAGA-30). A deleted version of this promoter sequencewas generated as well, with primers F70 (50-ACTATAGGCAAAA-TACGCAAC-30) and R81.5, to obtain a fragment from positions�900to +311. PCR fragments were purified and cloned into the pGEM-Teasy vector (Promega Corporation) and digested with HindIII andSmaI for cloning into the pBI101.1 binary vector. All plasmids weremaintained in the E. coli DH5a strain and DNA was purified fortransforming Agrobacterium tumefaciens, LBA4404 strain by electro-poration (60 mF, 200 V). Kanamycin-resistant colonies werescreened by PCR and Southern blot to verify that they containedthe promoter-GUS fusion and employed to transform A. thaliana

plants by the floral dip method [23]. Seeds were collected andgerminated in Gamborg’s B-5 (GIBCO BRL) agar media supplemen-ted with kanamycin (25 mg/ml) and cefotaxime (100 mg/ml). Atleast eight independent lines for each construction were selectedand T3 plants were employed for gene expression analysis.

2.5. GUS enzymatic assays

Histochemical detection of GUS activity was performed in T3plants using 5-bromo-4-chloro-3-indolyl-b-D-glucuronide asdescribed [24]. Tissues were destained with absolute ethanolbefore mounting for observation and photography in an OlympusCH30 microscope. For the fluorometric determination of GUS, totalprotein was extracted from plant tissue and enzyme activity wasdetermined with 4-methyl umbelliferyl glucuronide as substratein a Bio Rad VersaFluor fluorometer (560 nm excitation; 590 nmemission).

2.6. Semi-quantitative reverse transcription (RT)-PCR

In order to study ZmRBR2 expression in maize tissues by reversetranscription-PCR, a set of primers was designed based on the gene30-UTR. Forward Primer: 50-ATCTGGTTATTCCAAACAATCTG-30 andReverse Primer: 50-CAATGACTTCTCGCCTTCTCC-30. Such designallowed us to study the differential expression of the relatedZmRBR1 and ZmRBR2 genes, as the former transcript would yield a523-bp fragment while the latter would generate a 381-bp fragment(Fig. 6A). The ubiquitin transcript was reversed transcribed with thefollowing primers, ZmUBQF: 50-CACTTGGTGTTGCGTCTCAG-30/ZmUBQR: 50-CACCTCAAGGGTGATCGTCT-30, and used for RNAnormalisation. Semi-quantitative RT-PCR was performed with2 mg of total RNA from maize dehiscent pollen, roots, coleoptiles,leaf, stem, embryos axes, kernels and the whole seedling. cDNA wassynthesised using the ImPromII-Reverse Transcriptase (PromegaCorporation, Madison, WI) and PCR was performed with 0.2 mM ofeach primer and 1 cycle at 94 8C for 3 min, followed by 30 cycles of1 min at each one of the following temperatures: 94, 62 and 72 8C.The PCR products were resolved in a 1.2%-agarose gel, stained withethidium bromide and signal was quantified with a Fluor-SMultiImager (Bio-Rad). To analyse transcripts levels from pollentotal RNA, RT-PCR products were transferred onto a nylonmembrane and hybridised with a DIG-labelled cDNA probe (DIGHig Prime DNA labelling and detection starter kit, Roche).

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Hybridisation was carried out at 42 8C in 50% formamide andwashed at 68 8C. The cDNA probe corresponded to the 522-bpfragment that strongly hybridised with both DNA fragments.

2.7. Sequence alignment and promoter analysis

Sequence alignment was performed using ClustalW (http://seqtool.sdsc.edu/CGI/BW.cgi#!) [25]. Elements in the promoterregion were identified using the plant cis-acting regulatoryelements database (http://www.dna.afrc.go.jp/PLACE/signalscan.html) [26].

3. Results

3.1. ZmRBR2 gene structure

The DNA sequence of the 9.4 kb subclone (RB9000S) containedthe entire coding region of a RBR gene as well as 50and 30-flanking

sequences (GenBank Accession Number AJ279062). GC content ofthe ZmRBR2 gene was estimated and found to be 40% Primerextension analysis using total RNA from maize leaf and stemdisplayed two products with estimated sizes of 541 and 549 nt thatcorrespond to ZmRBR2 and ZmRBR1 transcripts respectively(Fig. 1A). Because primer P5 hybridised with both transcripts weconcluded that the 541 bp corresponded to ZmRBR2 as it is the lessabundant and our data will further show distinct transcript levelsof these genes. We mapped the transcription initiation site to aTAATAT hexanucleotide motif that is located 283 bp upstreamfrom the ATG codon (Fig. 1C).

Total DNA isolated from maize embryo axes was digested withBamHI, EcoRI, HindIII and SstI and the genomic blot was probedwith a 1534 bp probe obtained by digesting RB9000S with EcoRIand HindIII. The probe spanned exons 3–7 and under stringentconditions strongly hybridised to a 4.8 kb DNA fragment in BamHIdigestions that corresponded to the ZmRBR2 gene (Fig. 1B). A 2.3 kbfainter band was also observed that might belong to a related gene.

Fig. 1. Characteristics of the ZmRBR2 gene. (A) Transcription start site of ZmRBR2 gene. The products of the primer extension reaction using leaf (Le) and stem (St) total RNA

were resolved in a 4.5% polyacrylamide sequencing gel along with the sequencing reactions (A, C, G, and T). The arrow indicates the extended product corresponding to

ZmRBR2 RNA. (B) Southern blot analysis of maize genomic DNA (40 mg) digested with BamHI (B), HindIII (H), EcoRI (E) and SstI (S). A physical map of the RB9000S genomic

clone is shown in the bottom to account for the bands observed in the genomic DNA as well as the probe employed. (C) Nucleotide sequence around the major mapped

transcription initiation site mapped (black box) and the putative TATA box (gray box, white font). The nucleotide residues are numbered beginning with the major

transcription initiation site, and the nucleotides on the 50 side of residue 1 are indicated by negative numbers. The initiation codon is in bold characters and the arrow

indicates the position and length of oligonucleotide P5 used for the primer extension assay. A putative pollen-specific element AGAAA is enclosed in a gray box. C.

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Fig. 2. Comparative exon/intron structure of the ZmRBR2, OsRBR2 and AtRBR genes. Exons and introns are shown as black boxes and lines respectively. Exon sequences

showing high identity with ZmRBR2 exons are indicated. Translation initiation and termination sites are shown for the three genes.

Fig. 3. (A) The alignment of amino acid sequences of the N-terminal region of the three maize RBR proteins is shown. Identical residues are indicated with a dark background

and in the consensus line by an asterisk. The first amino acids corresponding to the A domain are indicated with a black bar. (B) Tree illustrating the relationship among

selected full-length RBR proteins. GenBank Accession Numbers are as follows: AtRBR (AAF79146), OsRBR1 (AAY23368), OsRBR2 (AAY23369), ZmRBR1 (CAA67422), ZmRBR2

(CAC82493), and ZmRBR3 (AAZ99092).

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Hybridisation with EcoRI-digested DNA yielded a 9 kb band and asecond band approximately 7 kb in size. Such sizes are consistentbecause the ZmRBR2 gene sequence contained only one EcoRI site,thus the probe would detect DNA fragments larger than 6 kb. Anintense, high-molecular-weight band was observed in the SstI-digested DNA that spanned the complete genomic clone isolatedhere, and probably a second RBR-related gene. Because thehybridisations and washing conditions were performed at highstringency, it is likely that the second band detected in theSouthern blot corresponded to ZmRBR1 as it has 80% identity withZmRBR2.

Alignment of the genomic sequence with the published maizeRBR cDNAs sequences allowed us to define an open reading framestarting at the ATG codon in position +283 and revealed that theZmRBR2 gene is composed of 16 exons and 15 introns summarisedin Fig. 2. Intron size varied from 64 to 651 bp and averaged a 37%GC content, and exon size varied from 42 to 1252 bp, although thelargest exon (15) contained the stop codon and a large sequencecorresponding to the 30-UTR sequence.

ZmRBR2 shared some common characteristics with other plantRBR genes as it contained the pocket A domain in a single exon (7)that is the largest exon in the translated region. This exon has a 47and 81% identity with exon 10 from AtRBR and OsRBR2 respectively.Also, the spacer region is contained in the next exon (8) that hashigh identity with exon 11 of the AtRBR and OsRBR2 genes, and thecoding region of pocket B was scattered among exons 9–12 (Fig. 2).In both monocot genes a large intron followed the exon-coding

pocket A domain whereas in AtRBR gene a short intron (94 bp) ispresent.

Sequence comparison allowed us to determine that the RBR

gene reported here corresponds to the partial ZmRBR2a

(AF007794) and ZmRBR2b (AF007795) cDNAs [18] as the 30

showed a higher degree of identity (97%) than with ZmRBR1

(88%) (X98923; AF007793) and with ZmRBR3 (34%) (DQ124423).Thus this isolated genomic clone allowed the analysis of the N-terminal region and most likely completing the set of three maizeRBR genes. Unlike the rest of the RBR genes that are composed of 17or 18 exons, ZmRBR2 coding region is contained in 15 exons andlacked the extended amino termini sequence that is present in theAtRBR and OsRBR2 genes. This sequence is contained in exons 2–6of these latter genes that code 159 amino acids that showed veryweak identity with the coding sequences of exons 1 and 2 ofZmRBR2 gene. Sequence alignment of the N-terminal region fromthe three maize RBR proteins showed high identity (88%) betweenZmRBR1 and ZmRBR2, and that both proteins are 142 amino acidshorter than ZmRBR3 (Fig. 3A). This high identity between ZmRBR1and ZmRBR2 suggest a gene duplication event that probablyoccurred after maize/rice speciation because the rice genome onlycontains two distinct RBR genes (Fig. 3B).

3.2. ZmRBR2 promoter

An AT-rich region was present upstream from the transcriptioninitiation site of the ZmRBR2 gene and a putative functional TATA

Fig. 4. Sequence of the ZmRBR2-gene promoter. Sequence numbering starts one nucleotide upstream of the mapped transcription initiation site. An AT-rich region with a

putative TATA box (gray boxes/white letters) is close to the transcription initiation site. A putative E2F element is indicated in bold and underlined and the four pollen-specific

motifs are shown in dark gray. Forward primers F71 and F70 employed to generate the respective promoter deletions are indicated with arrows.

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box (Fig. 4) was located 29 nucleotides upstream from thetranscription initiation site, which is consistent with other maizegenes [27]. A putative E2F/DP binding site (TTTGCCGG) wasdetected in the ZmRBR2 promoter region, 242-bp upstream of thetranscription initiation site (Fig. 4). The presence of this element isconsistent with other plant RBR genes; the OsRBR2 gene contains aTTTCCCGCG element 366-bp upstream from the start codon andOsRBR1 gene contains three potential E2F binding sites within thefirst 400-bp upstream of the start codon.

As a first approach to define its functional activity, a 1683 bp ofthe ZmRBR2 gene promoter was cloned into the pBI101 binaryvector that was used to transform A. thaliana plants. Organ- andtissue-specific expression patterns were studied histochemicallyat different developmental stages of T3 plants. During germination,prior to radicle emergence, low levels of expression were detectedin the cotyledons of germinating seedlings (Fig. 5A and B). GUSreporter activity was detected in the shoot apical meristem and inyoung cotyledons at 72 h of imbibition (Fig. 5C). Higher activity

Fig. 5. Histochemical localisation of ZmRBR2 promoter activity in transgenic A. thaliana plants transformed with construction F71. (A–C) Early stages of seedling development.

(D) Root tip. (E) Lateral root development. (F) Cotyledons. (G) Leaf primordia. (H) Rosette leaf. (I) Developing anthers. (J–L) Mature flowers during pollination. (M) Mature

pollen grains. (N) Ovules within the ovary.

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was detected in the vasculature of the cotyledons and remaineddetectable in 4-day-old seedlings as it gradually decreased in theexpanded cotyledon (Fig. 5F). No activity was detected in roottissue as neither root tips (Fig. 5D) nor developing lateral roots(Fig. 5E) were stained. During vegetative development, ZmRBR2

promoter activity was only detected in the vascular tissue of thehypocotyl (Fig. 5G), and was not detected in mature leaves or in thetrichomes (Fig. 5H).

Strong promoter activity was observed during early flowerformation, especially in the developing anthers (Fig. 5I–K). Maturepollen released from anthers showed an intense GUS activity(Fig. 5L and M) that was also detected in the stigma. Ovules withinthe ovary were also stained (Fig. 5N), but no activity was detectedpost-fertilisation during embryogenesis and seed development(data not shown).

In an attempt to explain the intense transcriptional activity inthese organs, we identified five copies of the pollen-specific motif(AGAAA). Four of the motifs were localised in the opposite strand at�1267,�1088,�778 and�452 bp from the transcription initiationsite and the fifth element was located in the transcribed strand atthe +57 position (Fig. 4). We generated A. thaliana transgenic plantstransformed with the 50-deleted version of the promoter (F70) thatwas 470 bp shorter (Fig. 4). These plants showed the same GUSexpression pattern (data not shown) as F71 plants, includingstrong activity in the anthers and pollen. Because this constructionlacked the two more distal AGAAA elements, we quantified GUS-specific activity in flower tissues using a fluorometric assay, anddid not find any significant differences between the floral tissuesfrom the two transgenic lines (257 � 20 pmol 4MU/min/mg proteinfor F70 vs. 259 � 39 pmol 4MU/min/mg protein for F71).

3.3. ZmRBR1 and ZmRBR2 gene expression

In order to study ZmRBR1 and ZmRBR2 differential expressionwe designed gene-specific primers based on the 30-UTR sequence.This region shows an 88% identity between the two transcripts, butRT-PCR with the primers employed allowed us to discriminate

both as the 30-UTR region of ZmRBR1 gene is 142 bp longer in theamplified region than the corresponding cDNA of ZmRBR2 (Fig. 6A)yielding two cDNA fragments that were resolved in agarose gels.We evaluated relative expression of both genes in seven differentmaize tissues: pollen (Fig. 6B), root, coleoptile, leaf, stem, embryo,kernel and the whole seedling. Both genes were expressed in alltissues (Fig. 6C) but ZmRBR1 transcript levels were consistentlyhigher than those of ZmRBR2 and approximately a 2:1 ratio wasobserved (Fig. 6D). These results are also consistent with primerextension data (Fig. 1A) that shows two distinct cDNAs withdifferent relative abundance). Because of the low amount of RNAobtained from pollen, the RT-PCR products were transferred to anylon membrane and hybridised with the DIG-labelled cDNAprobe but no quantification was performed.

4. Discussion

So far, three distinct RBR-cDNAs have been isolated from maizeand distinct lines of evidence suggest the existence of two closelyrelated genes (ZmRBR1 and ZmRBR2) plus a third more distantlyrelated gene, ZmRBR3 [19]. The rice genome contains two RBR

genes (OsRBR1 and OsRBR2) and two RBR cDNAs have been isolatedfrom wheat (TaRBR1 and TaRBR2p). According to the classificationproposed by Lendvai et al. [14], plant RBR proteins can becategorized in three distinct subfamilies; subfamily A comprises alldicot RBR proteins while subfamilies B and C contain monocot RBRproteins which are quite distinct. Subfamily B comprises ZmRBR3,TaRBR1 and OsRBR1 proteins and is actually closer to the dicotRBRs (subfamily A) as they share a 54–67% identity than tosubfamily C. The latter contains OsRBR2, TaRBR2p, ZmRBR1 andZmRBR2 and shows a 48–59% identity to the subfamily B members.ZmRBR1 and ZmRBR2 are closely related as they share 89% identitythus suggesting a recent gene duplication event that occurred afterrice/maize speciation. Completion of the full set of maize RBR genescontributes to extend the comparison of monocot genes with theirmammalian counterparts. In a similar fashion, human p107 andp130 genes are more closely related to each other than to RB [28].

Fig. 6. Transcript levels of ZmRBR1 and ZmRBR2 in various organs of maize plants. (A) Schematic representation of ZmRBR1 and ZmRBR2 30-UTR and oligonucleotide primers

used in RT-PCR. Arrows indicate the primers positions and the amplicon size for each transcript. (B) Agarose gel separation of RT-PCR products detected by Southern blot with

the specific probe. RNA was obtained from dehiscent maize pollen. (C) Agarose gel separation of RT-PCR products stained with ethidium bromide. Total RNA was obtained

from different tissues and used for RT-PCR: radicle from 48 h seedlings (R), coleoptile from 5-day-old seedlings (C), 10-day-old seedling (Se), leaf (L) and stem (St) from 20-

day-old plants, embryo (E), and kernel (K) at the milk stage. Band intensities were normalised with ubiquitin (UBQ) transcript. (D) Relative mRNA levels of ZmRBR1 and

ZmRBR2 were calculated using UBQ mRNA for normalisation and expressed as percentage of the maximum intensity. Error bars are �S.D. of two independent experiments.

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Expression patterns of the three human genes points to distinct butoverlapping roles, but gene redundancy has been proposed tocompensate when loss of RB function occurs as p107 is upregulated [29].

ZmRBR2 gene showed unique and conserved features with otherplant RBR genes. It is three-exons shorter than AtRBR and OsRBR2

and codes an 866 amino acid protein that is considerably shorterthan other plant RBR proteins such as AtRBR (1013 amino acids) orOsRBR2 (987 amino acids). Both, ZmRBR1 and ZmRBR2 lack anextended amino termini sequence that is present in other plantRBR genes. Conversely, ZmRBR2 shared other properties withdescribed plant RBR genes mainly that pocket A domain iscontained in a single exon (7) whereas the coding region of pocketB is distributed in four exons. A common feature of the twomonocot genes is that a large intron (651 bp for ZmRBR2 and926 bp for OsRBR2) follows the exon-coding pocket A domain whilein the dicot AtRBR gene a short intron (94 bp) follows thecorresponding exon. Intron position and number is generallyconserved in orthologous genes in plants as documented for the P-type ATPase ion pumps and recombination proteins in rice andArabidopsis [30] despite their diversification probably more than150,000,000 years ago [31].

The low GC content of the ZmRBR2 gene (40%), especially in theintrons (37%) places it as a GC-poor gene according to a recentclassification [32]. According to these authors, GC-poor genes alsohave shorter exons, averaging 300 bp, and in ZmRBR2, exon sizeaveraged 274 nucleotides. Although this distinction between GC-poor genes (40–55%) and GC-rich genes (55–75%) is very clear inGramineae and particularly in maize, its functional significance isnot clear, but several observations point out that housekeepinggenes are associated with GC-rich sequences and smaller intronsthat might facilitate active transcription. In contrast, GC-poorgenes with abundant and larger introns, favours alternativesplicing [32]. Interestingly, it has been shown that the ZmRBR2

transcript is alternatively spliced within the coding region to yieldtwo proteins with different C termini [18].

Promoter activity of the ZmRBR2 gene was analysed by stabletransformation of A. thaliana with two reporter fusions (F70 andF71). Although the fidelity of these promoter-GUS fusions cannotfaithfully reproduce the in vivo regulation of the gene, especially ina heterologous host [33], we found good correlation in most organsbetween GUS activity and gene expression data in both maize andA. thaliana.

By using a probe that cross-hybridised with both, ZmRBR1 andZmRBR2 genes, Ach et al. [18] showed that high transcript levels arepresent in various maize tissues, but particularly in the shoot apex.We also found high GUS activity in the shoot apex of A. thaliana

seedlings, thus suggesting an equivalent expression pattern in thisorgan in monocot and dicot plants. Microarray data shows aubiquitous expression of AtRBR through different organs but thehighest levels are observed in the shoot apex of 14-day-old plants[34]. AtRBR function in shoot apical meristem is to repress growthand to induce cells into the initial stages of differentiation [35].

Transcripts of ZmRBR1/RBR2 have been detected in maturemaize leaves [16,18] and AtRBR is expressed in rosette leaves atvarious developmental stages [34]. We found considerable GUSactivity in Arabidopsis leaf vascular tissue and leaf primordia thussuggesting the recognition of cis-elements in the maize promoter.These results are consistent with RBR function in A. thaliana duringleaf development as it restricts cell division and regulates theendocycle [9].

In contrast to the shoot apex and leaves, we could not establisha comparable tissue expression pattern in roots. While ZmRBR1/

RBR2 transcripts can be detected in maize roots by Northern blot[16,18] and AtRBR transcript by microarray experiments [34,36],

we could not detect GUS activity in the root tissue examined from7- and 10-day-old seedlings. These results point to the fact that cis-elements of the maize promoter are not recognized in Arabidopsis

root cells. A similar behaviour was reported for the promoterregion of a maize H3 histone gene in transgenic A. thaliana plantsbecause it did not drive GUS expression at the root tips, whereasthe homologous histone-3 gene promoter is capable of directingintense GUS staining in transgenic A. thaliana roots [37].

The high levels of GUS activity detected in floral organs,especially in anthers where pollen is formed, and in mature pollen,are consistent with RBR function at this developmental stage [11].Microarray analysis of microgametogenesis shows elevated RBR1

expression in uninucleate microspores that decrease in tricellularpollen and mature pollen grains [36]. Northern blot analysis showsa weak RBR signal in maize pollen [18] but we could detect bothZmRBR1 and ZmRBR2 transcripts in mature maize pollen grains byRT-PCR. Strong activity in pollen has been shown for othermonocot promoters such as the maize H3 gene which drives highGUS expression in transgenic A. thaliana pollen grains [37] or thewheat histone H3 promoter that induces strong GUS expression inpollen of transgenic rice [38]. The ZmRBR2 promoter contains threepollen-specific motifs (AGAAA) in the proximal region, one of themlocated at the 50-UTR that might be relevant in regulating RBR

expression. These elements might be well conserved in monocotsand dicots, as their function was originally described for thetomato lat52 gene and is transcribed in the vegetative cell duringpollen maturation. Its promoter contains three AGAAA upstreamelements and mutation of the element located at the �72 positioncauses a 10-fold reduction of the promoter activity in pollen [39].AtRBR promoter also contains such element in the proximal regionat �79 from the putative transcription initiation site. The relativeimportance of each element in the ZmRBR2 promoter could bediscerned with a more detailed deletion analysis on the twoproximal elements and the one located in the 50-UTR in malegametophytes. Genetic programs regulating gene expressionduring male gametogenesis are evolutionary conserved as similartranscripts are present in pollen of both mono- and dicotyledonousplants and the 50-UTR of various pollen mRNAs are important inregulating gene expression. ZmRBR2 contained hexanucleotideelements such as AGAAA (Fig. 1C) that are significantly overrepresented in the 50-UTR of pollen-expressed genes in dicots andmonocots. The functional significance of these elements isunknown, but their presence at the 50 end of the ntp303 geneaffect translation efficiency [40].

In the heterologous promoter–reporter system we foundequivalent expression patterns in most organs of A. thaliana plantsthat can be fully explained by the role of AtRBR duringdevelopment and gene expression data, both in maize andArabidopsis. There are various published examples in the literatureof plant promoters assayed in transgenic heterologous hosts thatpreserve their original tissue-specific expression [41,42]. A recentin silico study using local distribution of short sequences (LDSS)identified hexamer and octamer sequences within promoters of A.

thaliana and rice. Comparison of LDSS-positive sequences betweenthe monocot and dicot revealed shared promoter architecture anda moderate conservation of regulatory elements [43]. Although abioinformatic scrutiny predicts common elements between theAtRBR and ZmRBR2 promoters, only analysis of transgenic A.

thaliana plants expressing a homologous promoter-GUS fusioncould reveal the corresponding functionality of both promoters.

The fact that wheat, rice and maize, three monocot cereals,contain RBR proteins that belong to two distinct subfamilies supportthe concept of distinct and, maybe, additional roles of the RBpathway in monocotyledonous plants contrasting to dicotyledonousplants [14]. This complex network might involve interactions in the

M. Najera-Martınez et al. / Plant Science 175 (2008) 685–693692

Author's personal copy

monocot RBR pathway as it has been shown that ZmRBR3 expressionis controlled by ZmRBR1 through the E2F/DP heterodimer activity.ZmRBR1 is expressed ubiquitously in maize tissues, and duringendosperm development, transcript levels decreased graduallywhile ZmRBR3 transcripts at this stage, are detected in mitoticallyactive endosperm before falling to practically undetectable levels 11days-after-pollination [19]. The presence of ZmRBR2, an additionalmember of subfamily C of plant RBR proteins, with high identity toZmRBR1 brings up the question whether these two genes areredundant or carry out specific functions in plant development anddifferentiation, as well as the functional significance of thealternative splicing that occurs in ZmRBR2 transcripts. ZmRBR1/

RBR2 constitute the only duplicated genes within a subfamilyreported so far in plants and, although ZmRBR2 showed constitutiveexpression, transcript levels were lower than those of ZmRBR1 inthree tissues tested in this study. These results are consistent withthe difficulty in isolating a full-length ZmRBR2 cDNA [18]. Studies onthe spatial and temporal regulation of maize RBR genes coupled withfunctional analyses in maize mutants would help to determine thespecific function of each member of this family that would allow amore direct comparison between the monocot and dicot RBpathways.

Acknowledgements

This work was supported by research grants from CONACYT-3407PN to J.V.R. and BFU2006-5662 to C.G., and from CSIC-CONACYT exchange program. We thank P. Leon-Mejia and A.Guevara-Garcia for providing the LBA4404 A. tumefaciens strain,and R. Cruz-Ortega for critically reading the manuscript. Weacknowledge the technical help of L. Fabila-Ibarra, M.T. Olivera-Flores and M.J. Jimenez-Villalobos.

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