Gene 485 (2011) 53–62

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Gene

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Novel genes specifically expressed during the development of the male thalli and antheridia in the dioecious liverwort endiviifolia

Izabela Sierocka a, Aleksandra Rojek a,1, Dawid Bielewicz a, Wojciech Karlowski b, Artur Jarmolowski a,Zofia Szweykowska-Kulinska a,b,⁎ a Department of Gene Expression, Institute of Molecular and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 89 Umultowska Street, 61-614 Poznan, Poland b Laboratory of , Institute of and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 89 Umultowska Street, 61-614 Poznan, Poland article info abstract

Article history: In bryophytes (lower ), sex determination is manifested in the gametophyte generation by the Accepted 7 June 2011 production of egg- and sperm-forming gametangia. We identified four genes specifically expressed in the Available online 25 June 2011 male thalli of dioecious liverwort Pellia endiviifolia species B using RDA-cDNA method. These are: PenB_TUA1 coding for an α-tubulin family protein, PenB_Raba1/11 coding for a Rab family protein, PenB_HMG-box coding Received by A.J. van Wijnen for an HMG-box family protein and PenB_MT coding for an unknown transcript that contains an ORF of 295 amino acid residues. The expression of identified genes shows developmental and environmental regulation. Keywords: Antheridia PenB_TUA1 and PenB_Raba1/11 are expressed in the male thalli, regardless of whether they develop Liverworts antheridia. PenB_HMG-box and PenB_MT are exclusively expressed in the male thalli-producing antheridia Male gametophyte development while growing in the field. Moreover, two genes PenB_TUA1 and PenB_Raba1/11 are encoded only in the male Pellia endiviifolia genome of P. endiviifolia sp B. Our studies show for the first time the specific contribution of identified genes in RDA-cDNA the liverwort male gametophyte development. fi Sex-speci c gene expression In higher plants, correct regulation of α-tubulin and Rab family genes activity is essential for tip-focused membrane trafficking and growth of the male gametophyte. Thus these genes are critical to the reproductive success of these plants. HMG-box proteins bind DNA and may affect chromatin structure, promoting the assembly of nucleoprotein complexes that control DNA-dependent processes including . Our results show that genes connected with the gametogenesis processes are evolutionarily conserved from the liverworts — the oldest living land plants, to higher plants. © 2011 Elsevier B.V. All rights reserved.

1. Introduction with regard to many plesiomorphic features such as the cuneate apical cell, a thallus without the midrib, a spherical capsule and Pellia endiviifolia species B is a dioecious liverwort species massive seta (Crandall-Stotler and Stotler, 2000; He-Nygren et al., (phylum — , class , subclass Pellii- 2006). In P. endiviifolia sp B, as in all seedless embryophytes, sperm dae, order Pelliales, genus Pellia). The Jungermanniopsida comprise and egg cells develop within multicellular organs called antheridia over 80% of presently living liverwort taxa, in which Pellia species are and archegonia, respectively. These gametangia are formed exoge- recognized as the most basal lineage of the simple thalloid liverworts nously by the dedifferentiation of epidermal cells and develop on the thallus surface of the haploid male or female gametophytes (Paton, 1999; Schuster, 1992). These gametophytes are phenotypically Abbreviations: Aa, amino acid; aars, amino acid residues; bp, base pair; DP, identical until the sexual organs differentiate. As in all bryophytes, difference product; HMG-box, High Mobility Group box domain; kb, kilobase; nt, nucleotide; Rab, Ras related GTP-binding protein; RACE, rapid amplification of cDNA the sperm cells are bifllagelated motile cells, which are specialized to ends; RDA-cDNA, representational difference analysis of cDNA; Tua, α tubulin. swim in the water environment toward the egg cell for fertilization ⁎ Corresponding author at: Department of Gene Expression, Institute of Molecular completion (Renzaglia et al., 2000). Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 89 Dioecy is frequently found in the liverwort phylum — about two- Umultowska Street, 61-614 Poznan, Poland. Tel.: +48 61 829 57 66; fax: +48 61 829 59 49. thirds of Marchantiophyta are dioecious plants (Bell and Hemsley, E-mail addresses: [email protected] (I. Sierocka), [email protected] 2000). However, dioecy is rare among higher plants: only about 5% of (A. Rojek), [email protected] (D. Bielewicz), [email protected] the seed plant species develop male and female individuals (W. Karlowski), [email protected] (A. Jarmolowski), [email protected] (Tanurdzic and Banks, 2004). So far, the majority of researchers has (Z. Szweykowska-Kulinska). focused on the seed plants to study mechanisms involved in plant sex 1 Present address: University of Medical Sciences in Poznan, Faculty of Medicine I, 2nd Chair of Pediatrics Department of Pediatric Endocrinology and Rheumatology determination. For example, members of the genus Silene represent Molecular Endocrinology Laboratory, 27/33Szpitalna Street, 60-572 Poznan, Poland. an important model system to study the early stages of sex

0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.06.012 54 I. Sierocka et al. / Gene 485 (2011) 53–62 chromosome evolution. Investigations have been focused mainly on 2.2. Representational difference analysis of cDNA (RDA-cDNA) the studies of dioecious species with large heteromorphic sex chromosomes (XX/XY sex-determining system). Determination of RDA-cDNA analysis was performed according to Hubank and male- or female individuals in Silene latifolia was found to be Schatz (1994) and Kwasniewski and Szarejko (2006) with several depended on the role of the Y chromosome. The genes present on modifications (Ausubel et al., 2003). Male gametophyte-producing the Y chromosome are responsible for the male-sex promotion, anther antheridia was used as the TESTER material for RNA isolation and development and male fertility. Genes necessary to prevent the female gametophyte-producing archegonia as the DRIVER. Total RNA female sex organs development are localized on this chromosome for RDA-cDNA analysis was isolated using hot phenol extraction (Koizumi et al., 2007; Mrackova et al., 2008; Nicolas et al., 2004). A method (Boutla et al., 2002) or, alternatively, a single-step method substantial amount of data is also available on the genetics of described by Chomczyński and Sacchi (1987), and modified by Pant reproductive organ formation and function in such plants as (Pant et al., 2009) was followed. The poly(A)+ RNA was isolated with Arabidopsis thaliana, Zea mays, or Lilium longiflorum including germ- the PolyATract® mRNA Isolation System (Promega) and double line cells and their differentiation (Borg et al., 2009; Drews et al., stranded cDNA (ds-cDNA) was synthesized using the cDNA Synthesis 2010). System (ROCHE). Oligonucleotides utilized in RDA-cDNA were as In lower plants, like bryophytes, genetic processes responsible for published in Hubank and Schatz (1994). 1.5 μg of the TESTER and the the transition from vegetative to reproductive stage are largely DRIVER ds-cDNAs was employed to generate amplicons. Four rounds unknown. Liverworts are considered by many researchers as the of subtractive hybridization/amplification were performed, using the oldest lineage of bryophytes (He-Nygren et al., 2006). Due to their quantitative TESTER to DRIVER ratios as follows: 1:100 for the first unique position in the evolution of land plants, liverworts may serve round, 1:800 for the second round, and 1:400 000 for the next two as models to investigate the molecular basis of mechanisms involved rounds. Hybridization was carried out at 67 °C for 48 h. After the in sexual reproduction. For example, the first plant dimorphic sex fourth round of hybridization (DPIV) the RDA-cDNA products were chromosomes were described in the dioecious representative of separated by electrophoresis in 1.5% agarose gel, recovered using the liverworts, Sphaerocarpus texanus, in which the larger X chromosome QIAquick Gel Extraction kit (Qiagen), and cloned into the pGEM-T- is responsible for female gametophyte development and the smaller Y Easy vector (Promega). DNA was sequenced using the BigDye chromosome is responsible for male gametophyte development Terminator v3.1 cycle sequencing kit (Applied Biosystems) according (Allen, 1917). A similar situation was shown in the dioecious to the manufacturer's protocol. Sequences were determined with the Marchantia polymorpha, where X and Y chromosomes have been 3130xl Genetic Analyzer (Applied Biosystems). characterized as cytologically different (Bischler, 1986; Nakayama et al., 2001; Newton, 1984). In both species sex chromosomes are 2.3. RT-PCR analysis markedly smaller than autosomes and determine the sex of haploid gametophytes. In recent years advanced methods of molecular A reverse transcription assay was carried out using Oligo-dT(15) biology have been used to study the genome structure of M. (Novazyme) and SuperScript III Reverse Transcriptase (Invitrogen) polymorpha. The Y chromosome of this liverwort was the first to be according to the manufacturer's instructions. To identify fragments of sequenced in plants and the resolution of its structure provided a expressed genes (selected as DPIV products), six pairs of Forward and detailed view of a male-sex chromosome in a haploid organism. Out of Reverse oligonucleotide primers were designed (Supplementary 64 genes found on the M. polymorpha Y chromosome 14 were Y- materials, Table S1). Reactions were standardized to P. endiviifolia sp chromosome specific. Their expression was detected exclusively in B ACTIN1 expression level (accession no. DQ100290). The following the male reproductive organs that suggest their involvement in male thermal cycle program was used for all semiquantitative RT-PCRs: 30 reproductive processes (Yamato et al., 2007). M. polymorpha belongs cycles of 95 °C for 5 min; 94 °C 30 s, 58–64 °C for 30 s, 72 °C for 1 min; to class , which comprises liverworts with the most and an extension at 72 °C for 10 min. complex organization of thalli and sex organs (Bell and Hemsley, Real-time PCRs were performed using a 7900HT Fast Real-Time PCR 2000). This classification reflects their relatively younger evolutionary System and Power SYBR Green PCR Master Mix (Applied Biosystems), age when compared to liverwort from the Jungermanniopsida class. cDNA, and gene-specific primers (200 nM each) in a final volume of 10 μl. In this study, we identified four genes and their transcripts that are The following thermal profile was applied for all real-time PCRs: 95 °C for specifically expressed in the male thalli of the liverwort P. endiviifolia 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. After each real-time sp B, a representative of the most basal lineage of the simple thalloid PCR run, dissociation curve analyses were performed. The results were liverworts. In addition, we determined that the expression of two analyzed with the SDS 2.2.3 software (Applied Biosystems). Ct values for identified genes is developmentally and environmentally regulated, all transcripts were normalized to the ACTIN1 Ct value. Fold change values and is detectable only in the male individuals producing antheridia were calculated using the comparative ΔΔCt method. while growing in the natural habitat. 2.4. Quantification of two alternatively spliced mRNA isoforms of PenB_HMG-box gene 2. Materials and methods Total RNA was isolated from P. endiviifolia sp B. male thalli-producing 2.1. Plant material antheridia collected in the third season (2008) from the natural habitat. Two real-time PCR reactions were performed to detect the specific Female and the male thalli of P. endiviifolia sp B (Szweykowski et mRNA isoforms of PenB_HMG-box gene with the use of isoform-specific al., 1995) producing sex organs were collected from Kopanina, primers (Supplementary data, Table S2). The two reactions had Poznan, Poland (Herbarium number 40228 in POZW) during three equivalent efficiencies that allowed calculation of the percent abundance seasons: 1) September–December 2006, 2) 2007, and 3) 2008, of two mRNA isoforms [100×HMGlong/(HMGlong+HMGshort)] respectively. The in vitro collection was started from each sample (Supplementary data, Fig. S4). that was first propagated on mineral medium (Fiedorow and Szweykowska-Kulińska, 1998) and subsequently on half-strength 2.5. RACE and genome walking experiments Gamborg medium (Sigma). Plants from the axenic culture were grown under continuous white light provided by fluorescent lamps at In order to obtain full length cDNA sequences rapid amplification 21–23 °C. of 5′ and 3′ cDNA ends (5′/3′ RACE) technique was performed using I. Sierocka et al. / Gene 485 (2011) 53–62 55 the SMARTTM cDNA Amplification Kit (Clontech) according to the 3.2. Isolation and characterization of genes specifically expressed in the manufacturer's instructions. Full genomic sequences of selected RDA- male P. endiviifolia sp B gametophytes and their transcripts cDNA fragments were established by genome walking method with the GenomeWalker™ Universal Kit (Clontech) and DyNAzymeTM EXT To obtain the full-length cDNA and genomic clones for the four DNA Polymerase (Finnzymes). Genomic DNA for genome walking selected RDA-cDNA fragments, the 5′/3′ RACE and genome walking experiments was isolated using the DNeasy Plant Mini Kit (Qiagen). experiments were performed. We identified 5′ and 3′ cDNA ends of the four transcripts studied. In all cases primers used for 5′ and 3′ RACE were designed based on sequences obtained in the RDA-cDNA 2.6. Bioinformatic analysis experiment. To demonstrate that the longest 5′ and 3′ transcript ends belonged to the same transcript molecule, the RT-PCR for all four Database searches of the nucleotide and amino acid sequences transcripts was carried out using primers designed for the 5′ and 3′ were performed through an NCBI/GenBank/Blast website. In order to ends of the longest RACE products. For all transcripts the expected determine the homology between the predicted proteins encoded by products were obtained when RNA isolated from the male gameto- selected genes CLUSTALW2 program was used. The alignments were phyte producing antheridia was used. The full-length cDNAs are fi visualized with BOXSHADE 3.21 program. The search for speci c shown in Fig. 3 (lane 3 in all panels). amino acid sequences was made with MotifScan (http://myhits.isb- Initially, genome walking studies were carried out using primers sib.ch/cgi-bin/motif_scan), InterProScan (http://www.ebi.ac.uk/ designed for the four selected RDA-cDNA fragment sequences. Tools/InterProScan/)andSMART(http://smart.embl-heidelberg. Consecutive genome walking steps were performed until the distal de/) programs. The subcellular location of predicted amino acid 5′ and 3′ cDNA end sequences were found within the genomic sequences was assigned with PSORT (http://psort.imsekundu- sequences. To demonstrate that the identified genomic fragments tokyo.ac.jp/form.html), TargetP 1.1 (http://www.cbsekunddtu.dk/ were parts of the same gene we carried out PCR for all four genes services/TargetP/) and ProtComp (http://linux1.softberry.com/berry. using primers designed to the 5′ and 3′ ends of the longest RACE phtml?topic=protcompplandgroup=programsandsubgroup=proloc) products using genomic DNA isolated from the male thalli as a programs. The computation of various physical and chemical properties of template. The full-length genes amplified using PCR and separated proteins was assessed with ProtParam tool (http://expasy.org/tools/ electrophoretically are shown in Fig. 3 (lane 4 in all panels). The – protparam.html). The exon intron structures of selected genes were alignment of cDNA and genomic nucleotide sequences allowed us to established using FGENESH program (http://linux1.softberry.com/berry. identify gene structures, including exon/intron content, and untrans- fi phtml?topic=fgeneshandgroup=programsandsubgroup=g nd), and lated regions (UTRs). Moreover, the blast analysis of the full-length the alignment of cDNA and corresponding genomic sequences. cDNA sequences allowed us to identify the genes. We identified them as: α-tubulin (PenB_TUA1), Rab a1/11 (PenB_Raba1/11), and HMG- 2.7. GenBank accession numbers box protein (PenB_HMG-box). One gene, PenB_MT,showedno similarity to sequences registered in the public databases, and was — Sequences of full length cDNA and genomic sequences of selected designed as a Mysterious Transcript MT. The two cDNA fragments, genes were submitted to GenBank (accession numbers: HQ634388 – 273 nt and 229 nt, represent fragments of PenB_TUA1 and PenB_Ra- HQ634391, HQ634384 – HQ634387). ba1/11genes, respectively, while the remaining two cDNA fragments, 243 nt and 193 nt, represent fragments of PenB_MT and PenB_HMG- box genes. 3. Results 3.2.1. Molecular characterization of the PenB_TUA1 gene 3.1. Isolation of cDNA fragments of genes specifically expressed in the The structures of the PenB_TUA1 gene and its transcript are male P. endiviifolia sp B gametophytes using RDA-cDNA approach summarized in Fig. 4A. The mRNA is 2216 nt long, with the ORF of 1344 nt, a 235 nt-long 5′UTR, and 633 nt 3′UTR. Within the gene we The RDA-cDNA technique was employed for dioecious liverwort P. predicted one polyadenylation signal ACTAAA located 437 nt endiviifolia sp B to identify genes involved in the development of male downstream from the stop codon TAG. The PenB_TUA1 gene is thalli and antheridia. cDNAs obtained from the liverwort thalli 4541 bp long and contains five exons (348 bp, 215 bp, 200 bp, 202 bp, collected from the natural environment during two seasons (2006 944 bp, respectively) and four introns of the U2-type (309 bp, 602 bp, and 2007) were used in four rounds of subtractive hybridization. 695 bp, 1026 bp, respectively). The ORF of PenB_TUA1 encodes a 448 cDNA obtained from RNA isolated from the male gametophytes aa-long protein with a calculated molecular mass of 49.5 kDa and a producing antheridia was used as the TESTER and cDNA obtained predicted pI of 4.99. PenB_TUA1 protein shows a high degree of from RNA isolated from the female gametophytes producing identity with known plant α-tubulin family members: it has 97% archegonia, as the DRIVER. Although initial male and female identity with α-tubulin from Physcomitrella patens, α-tubulin 6 from amplicons were visually indistinguishable from each other (Fig. 1A), Z. mays, α-tubulin 3 and 5 from A. thaliana, and 95% identity with α- a stepwise reduction of complexity of the products in successive tubulin 1 from Oryza sativa (Supplementary data, Fig. S2). MotifScan, subtractive hybridization rounds was clearly visible (DPI–DPIV, InterProScan, SMART analyses indicated two globular domains Fig. 1B). The DPIV products were obtained as distinct bands ranging characteristic for α-tubulins that cover almost entire protein from 200 to 300 bp in size (Fig. 1B). These DPIV cDNA fragments were sequence: N-terminal with GTPase activity [3–245 Aars] and C- cloned and sequenced. Overall we obtained six individual sequences terminal tubulin/FtsZ family domain [243–393 Aars]. Moreover, NCBI encoding different transcripts. To confirm whether these fragments Conserved Domain Database search identified the nucleotide binding representing six distinct genes of P. endiviifolia sp B were specifically domain and 20 conservative amino acid residues responsible for expressed in the liverwort male thalli, six primer pairs were designed interactions of the α- and β-tubulins interface. The subcellular based on obtained DPIV sequences (Supplementary data, Table S1). A localization of the PenB_TUA1 protein is most likely cytoplasmic, as semiquantitative RT-PCR analyses were performed with the same predicted by various algorithms. RNA template the one used for RDA-cDNA experiment. The expression in male gametophytes was confirmed for four out of six isolated DPIV 3.2.2. Molecular characterization of the PenB_RABa1/11 gene products (Fig. 2, line 2). More importantly, these fragments were not The structure of the PenB_Raba1/11 gene and its transcript is present in the cDNA derived from the female thalli (Fig. 2, line 1). diagrammed in Fig. 4B. The mRNA is 1330 nt in length, with the ORF of 56 I. Sierocka et al. / Gene 485 (2011) 53–62

Fig. 1. RDA-cDNA amplicon analysis conducted on 1.5% agarose gels. (A) cDNA of the TESTER (M-male in line 2) and the DRIVER (F-female in line 3) amplicons are shown. Two types of molecular weight markers were used: 1 kb ladder in line 1, and 100 bp ladder in line 4. (B) Differential products (DP) after first (DPI), second (DPII), third (DPIII), and fourth (DPIV) round of subtractive hybridization are presented. The 100 bp ladder is presented in lane 1. mentary data, Fig. S3). MotifScan, InterProScan, SMART analyses confirmed that the predicted protein is a Rab family protein. MotifScan indentified a conserved domain characteristic for the Rab1 protein family covering almost the whole protein sequence [8–215 aars]. The PenB_Raba1/11 aa sequence contains guanine nucleotide-binding domains and an effector-binding domain (Supplementary data, Fig. S3). These include the following subdomains: G1 (P-loop, GDSGVGKS, 21–27 aars) involved in Mg+and phosphate binding; G3 (WDTAGQ, 57–61 aars), where DTAG interacts with γ-phosphate of GTP; G4 (GNKSD, 115–119 aars), where NKXD is the guanine specificity region; G5 (ETSAL, 150–155 aars), where ETSA interacts with the D residue in the NKXD. All these motifs together are responsible for GTP and GDP binding, and GTP hydrolysis. The biological function of small G proteins strongly depends on post-translational modification. The Ras, Rho, and Rab families have a C-terminal sequence, which undergoes post- translational modification, and is essential for cellular localization and interaction with different effectors. Most Rab proteins have two Fig. 2. Semiquantitative RT-PCR analyses of four cDNA fragments identified in RDA- cysteine residues embedded within the prenylation motif (XXCC, cDNA approach, using RNA template prepared for RDA-cDNA experiment from the XCXC, CCXX, CCXXX, XCCX, where X represents any aa). The cysteine female (lane 1) and male (lane 2) thalli producing sex organs. The results of PCR residues are modified by geranyl moieties (Agarwal et al., 2009). We reaction without template are shown in lane 3. The 100 bp ladder is in lane 4. identified the prenylation motif within PenB_Raba1/11 protein (GCCS, 213–216 aars, Supplementary data, Fig. S3). The PenB_Raba1/11 651 nts, a 272 nt-long 5′UTR, and 409 nt 3′UTR. Within the gene we protein was predicted to be most likely located in the cytoplasm, predicted one polyadenylation signal AATAA located 128 nts down- however, the prenylation motif strongly suggests its attachment to the stream from the stop codon TGA. The PenB_Raba1/11 gene is 2485 bp membrane. long and contains two exons (490 bp, 845 bp, respectively) and one intron of the U2-type (1150 bp). The ORF of PenB_Raba1/11 encodes a 3.2.3. Molecular characterization of the PenB_MT gene 217 aa long protein with a calculated molecular mass of 24.037 kDa The structure of the PenB_MT gene and its transcript is summa- and a predicted pI of 6.33. PenB_Raba1/11 shows a high degree of rized in Fig. 5A. MT transcript is 2689 nt long, with the ORF of 885 nts, similarity with known plant Rab a1 or Rab11 family members: it has a 1197 nt long 5′UTR, and 605 nt long 3′UTR. Within the gene we 92% identity with predicted GTPase from Selaginella moellendorffii, 83% predicted one polyadenylation signal AATAA located 562 nt down- identity with AtRabA1f from A. thaliana, 82% identity with hypothetical stream from the stop codon TAA. The PenB_MT gene is 3875 bp long GTP-binding protein from O. sativa, 77% identity with Rab11b protein and contains two exons (2803 bp and 607 bp, respectively) and one from Z. mays, and with Rab11/RabA protein from P. patens (Supple- intron of the U2-type (1185 bp).The ORF of PenB_MT encodes 295 aa-

Fig. 3. The full-length genes (lane 4 in panels A–D) and their transcripts (lane 3 in panels A–C plus lanes 3a and 3b in panel D) are analyzed on 1% agarose gels. PenB_TUA1 (A), PenB_RABa1/11 (B), PenB_MT (C) and PenB_HMG-box (D). The PCR reaction without template is shown in line 2. 1 kb+ ladder is in line 1 in panels A–D. I. Sierocka et al. / Gene 485 (2011) 53–62 57

Fig. 4. Schematic representation of the PenB_TUA1 (A) and the PenB_RABa1/11 (B) genes and their transcripts. Exons are represented by boxes, introns by lines; dark gray boxes denote 5′ and 3′ UTRs; light gray boxes denote the coding sequence. The dashed box within 3′UTR (A) and within the second exon (B) represents the fragments derived from the RDA-cDNA experiment. The black triangle indicates a bioinformatically-identified polyadenylation signal. long protein with a calculated molecular mass of 30.709 kDa and a 3′ acceptor site within the third intron is used — a shorter transcript predicted pI of 6.70. Our public database searches did not identify any (758 nt) is generated that contains 3′ UTR of 129 nts. Alternatively, similarity to known proteins. Analysis with InterProScan program when the distal 3′ acceptor site is recognized, a longer transcript showed the presence within N-terminal region of predicted protein a (1164 nt) is generated, with very long (535 nt) 3′UTR. The observed putative signal peptide [1–22 Aars] overlapping transmembrane alternative splicing events do not interfere with the coding sequence, domain [15–35 Aars] (Fig. 5B). ProtParam program established that which in both cases is 360 nt long. Both isoforms have an identical 5′ the most frequent amino acid residues are leucine (17.3%), threonine UTR region (266 nt). Within the longer mRNA isoform we predicted (11.9%), proline (9.8%), asparagine (9.2%), and serine (8.8%). one polyadenylation signal ATAAA that is located 80 nt downstream However, there are no conserved domains or motifs characteristic from the stop codon TAA. Although the shorter mRNA isoform is also for leucine-, threonine- or proline-rich proteins. Analysis with polyadenylated (129 nt downstream from the stop codon), the different bioinformatic tools showed that the predicted protein is polyadenylation signal within 3′UTR was not identified. Real-time located in the plasma or peroxysome membrane. PCR reactions were performed to determine the amount of two alternatively spliced mRNA isoforms of PenB_HMG-box gene tran- 3.2.4. Molecular characterization of the PenB_HMG-box gene script. As shown in Fig. 6B, the dominant isoform is the mRNA with the The PenB_HMG-box gene is 2412 bp long and contains four exons shorter 3′UTR, while the other one with the longer 3′UTR appears to (342 bp, 212 bp, 75 bp, 584 bp, respectively) and three introns of the be less abundant. This may indicate that the mRNA isoform with the U2-type (582 bp, 185 bp, 432 bp). The structure of the PenB_HMG-box longer 3′UTR results from the less frequent splicing event or gene and its two mRNA isoforms, is summarized in Fig. 6A. Two mRNA alternatively, that it is less stable than the shorter one and is isoforms are the result of alternative splicing within the third intron degraded. Bioinformatic database search was performed to find where two alternative 3′ acceptor sites are used. When the proximal possible AU-rich elements, potential signal sequences for transcript

A

B PeB_Mt/1-64 1 MHQSEAMKIFMKSVLLVAAIAGQLLNSSHIAQAAGLTLPPILDPIINPINNLTSPILDPLNNLT PeB_Mt/65-128 65 TPLLSPITNLTSPILDPLNNLTSAPIVGLNLTTVLGSLNLTAGTPALNLSTILGPLNLTAGTGL PeB_Mt/129-192 129 NLSNIIGPWNNLTGPLQTILSNFTGPASNLTVATNVTVTVTLNLPSTMLNLASVFPNGMTIVPL PeB_Mt/193-256 193 GMTEVSISLRSLQGTNLSTVPVQFMVPVISYLNFPVSGSAGVTLDIAPGTKLAITGLTTPITRL PeB_Mt/257-295 257 FFGVSWQTFPQLTNVLISKILPGSSQPVPLYNFAVPALM 295

Fig. 5. Schematic representation of the PenB_MT gene and its transcript (A). Amino acid sequence of predicted PenB_Mt protein (B). The boxed region indicates a putative signal peptide, and the underlined region — a putative transmembrane domain. All designations are the same as in Fig. 4. 58 I. Sierocka et al. / Gene 485 (2011) 53–62 A

B

C

Va-a_NBP/1-36 1 ------MPKAAAKRGAGEKKAKRAKKDPNAPKRGLSAYMFFA Chg_conhypP/1-36 1 ------MPKAAKSRSGKAEKKTRQKKDPNAPKRGLSAYMFFA Ad_NBP/1-35 1 ------MPKEKTTRKTKVRGEKK-KKDPNAPKRGLSAYMFFA Vc_hypp/1-35 1 ------MPKAAKEKPAAAKREKK-VKDPNAPKKPLGAYMWFC PeB_HMGbox/1-60 1 MGKATLKTKGKSGSGRGVTKASSSRCRSSSKRKGKEEKRIRKVKDKRRPKRALGPYMYFC Pp_pHMGbox/1-33 1 ------KAISSAKR---AIRKLRKQERAWRPKRAIGPYMFFC consensus 1 ...... **... .**.*

# Va-a_NBP/37-93 37 NEQRENVREENPGISFGQVGKLLGERWKALNEKQRGPYEAKAVADKKRYEDEKAAYN--- Chg_conhypP/37-93 37 NEQRDNVREENPGVSFGQVGKILGERWKALSDKQRAPYEAKAAADKKRYEDEKQAYN--- Ad_NBP/36-92 36 NEQRDNVREENPGISFGQVGKVLGERWKALNEKQRAPYEAKAAADKKRYEDEKASYNARR Vc_hypp/36-92 36 KDMRERVKAENPGMSVTDIGKRLGELWKEVSEEDKKKYLKQAEDDKERYNKEAAAYN--- PeB_HMGbox/61-117 61 KDQRKEIQEQNPTMSFGDIGRVLGSQWGKLNEKEKQKYIRKAQTDKRRYVKEMKRYK--- Pp_pHMGbox/34-90 34 KDQHANVTADNPSIPFTEIGKILGAQWQQMNEKDKKPYIKRSEVDKKRYEKELKRCK--- consensus 61 **...... **...... *..**..*...... *.... .**.**. *.....

Va-a_NBP/94-102 94 -AEAEDDESA 102 Chg_conhypP/94-96 94 -VSV------96 Ad_NBP/93-105 93 KAQDDDDESS 105 Vc_hypp/93-94 93 -KE------94 PeB_HMGbox/118-120 118 -PRY------120 Pp_pHMGbox/91-92 91 -LK------92 consensus 121

Fig. 6. Schematic representation of the PenB_HMG-box gene and its two mRNA isoforms (A). All designations are the same as in Fig. 4. Real-time PCR analysis for the quantification of two alternatively spliced isoforms of PenB_HMG-box gene in P. endiviifolia sp B. male thalli-producing antheridia (B). Material was collected in the third season (2008) from the natural habitat. Calculation shows mean±SD from three technical replicates. Amino acid sequence alignment of the HMG-box domain-containing proteins from V. albo-atrum (Va- a), C. globosum (Chg), A. dermatitidis (Ad), V. carteri (Vc) P. patens (Pp) (GenBank Acc. Nos. XP_001756119, XP_001229234, EEQ83979, XP_002957151, XP_001758727, respectively) and P. endiviifolia sp B (PeB) (C). Highly-conserved amino acid residues are in black, conserved substitution of amino acid residues are indicated in gray, white marks non-conserved amino acid residues. Hyphens mark deletions of amino acids. The HMG-box domain is boxed. Well-conserved Phe and Val residues that may potentially interact with DNA are indicated by the gray star symbol. An invariant Trp residue within the HMG-box domain is indicated by pound mark (#).

degradation. However, none of presently known AU-rich elements protein has 48% identity with a predicted protein with an HMG-box was found within the longer 3′UTR region. It cannot be excluded that domain from P. patens and a hypothetical protein V. carteri, and 42– transcripts of P. endiviifolia sp B possess signal sequences responsible 46% identity with nucleosome-binding proteins from fungi. MotifS- for degradation of mRNAs not yet characterized and specific for can, InterProScan and SMART analyses indicated an HMB-box domain liverwort phylum. [49–117 aars] and a bipartite nuclear localization signal [32–49 and The ORF of the PenB_HMG-box encodes 120 aa protein with a 50–65 aars]. Cellular localization prediction analyses confirmed calculated molecular mass of 14.12 kDa and a predicted pI of 10.72. A nuclear localization of the predicted PenB_HMG-box protein. The blastp search of the NCBI database indicated that the predicted HMG-box domain binds linear DNA in a non-sequence-specific way protein shows moderate homology to HMG-box domain-containing with moderate affinity. The aa residues responsible for primary and proteins from the moss P. patens, the algae Volvox carteri f. nagariensis secondary DNA interactions are conserved phenylalanine and valine, and several fungi species: Ajellomyces dermatitidis, Verticillium albo- which are also preserved in the PenB_HMG-box protein (Fig. 6C) (He atrum, and Chaetomium globosum (Fig. 6C). The PenB_HMG-box et al., 2000; Klass et al., 2003; Masse et al., 2002). Moreover, the I. Sierocka et al. / Gene 485 (2011) 53–62 59 invariable tryptophan residue that is important for the correct HMG- real-time PCR carried out for all four investigated gene transcripts box fold (Thomas and Travers, 2001) is also present in the established the relative transcript levels in each thalli tested. PenB_HMG-box (Fig. 6C). The PenB_HMG-box protein, while showing Transcripts of PenB_TUA1 and PenB_RABa1/11 genes accumulated in homology to other HMG-box proteins, differs from them by having an three different types of the male thalli of the liverwort with the extended 24 aa long hydrophilic N-terminus. highest level in the male gametophytes cultured in vitro. It should be noted that P. endiviifolia sp B thalli cultivated in vitro very rarely 3.2.5. PenB_TUA1 and PenB_Raba1/11 genes are present only in the male develop sex organs, and even when these are produced they do not genome of P. endiviifolia sp B mature. This observation indicates that axenic conditions of growth To investigate whether the identified genes are present in the are not sufficient for proper liverwort thalli development and that an genomes of both female and male individuals, or are present environment has a contribution in gene transcription regulation. The exclusively in male genomes, numerous PCR reactions were per- observed differences of transcript levels of PenB_TUA1 and Pen- formed using templates genomic DNA isolated from the male and B_RABa1/11 genes between three different thalli may reflect disrup- female thalli grown in vitro and in natural habitat. Surprisingly, tions in transcriptional regulatory mechanisms. The higher transcripts PenB_TUA1 and PenB_Raba1/11 genes amplified only from the male ratios in in vitro cultivated male gametophytes may be the result of genomic DNA template (Fig. 7, lane 2), with no amplification from the lack of specific agent(s) from natural environment that regulate(s) female genomic DNA (Fig. 7,lane1).Thisconfirms that the their proper level of transcriptional expression. In the case of the two corresponding genes are present only in the male genome. PenB_MT remaining genes, qPCR confirmed that the transcripts of PenB_MT and and PenB_HMG-box genes were amplified both from the male and PenB_HMG-box showed accumulation only in the male thalli of P. female genomic DNA, indicating that these genes are present in both endiviifolia sp B grown in the natural environment during antheridia male and female genomes (Fig. 7, lines 1, 2). development. The absence of their expression in axenic culture strongly suggests that in vitro conditions are not sufficient for the 3.3. Expression of identified genes is developmentally and environmentally proper development of P. endiviifolia sp B thalli and for completion of regulated in the male P. endiviifolia sp B gametophytes sex organs development. We conclude that PenB_TUA1 and PenB_RABa1/11 genes show To investigate the possible developmental and environmental male-specific expression in P. endiviifolia sp B gametophytes, while regulation of identified genes expression analyses were performed by PenB_MT and PenB_HMG-box genes exhibit much more restricted semiquantitative RT-PCR. Additionally, real-time PCR approach was developmental expression regulation and are expressed in the male employed to quantify the amounts of transcripts in different individuals grown only in the natural environment during antheridia developmental stages of the male individuals grown under various production. conditions. RNA was isolated from different types of the female and male P. endiviifolia sp B gametophytes. These were as follows: (i) the 4. Discussion female thalli without archegonia cultivated in vitro, (ii) the female thalli producing archegonia collected from the natural habitat, (iii) 4.1. The identification of genes specifically expressed in the dioecious the male thalli with and without antheridia cultivated in vitro, and (iv) liverwort P. endiviifolia sp B the male thalli producing antheridia collected from the natural habitat (we were not able to discriminate between the male and female thalli Since the transcription of most genes results in specific protein in nature when liverwort do not produce sex organs). Semiquanti- production and function, changes in the transcriptome reflect changes tative RT-PCRs showed that all four genes are specifically expressed in in the biochemical processes ongoing in specific type cell, tissue, themalegametophytes(Fig. 8A). Two genes, PenB_TUA1 and organ, or even organism. Thus studying transcriptional changes may PenB_RABa1/11,showedspecificexpressionbothinthemale provide novel insights into many biological processes at the molecular gametophytes cultured in vitro (without or with antheridia) and in and physiological level. The relative simplicity and sensitivity of the the male gametophytes producing antheridia and grown in the RDA-cDNA technique allowed us to examine differences in gene natural habitat (Fig. 8A, lines 3, 4, 5). Moreover, these transcripts were expression between the male and female gametophytes producing not detected in the female thalli (Fig. 8A, lines 1, 2). Most interestingly sex organs of the dioecious liverwort P. endiviifolia sp B. We selected the remaining two genes, PenB_MT and PenB_HMG-box, showed four genes specifically expressed in the male liverwort thalli of P. specificexpressiononlyinthemalegametophytes producing endiviifolia sp B. Most importantly this technique allowed us to antheridia and grown in natural habitat (Fig. 8A, line 5).Quantitative identify new genes and their transcripts without any knowledge of the genome and transcriptome sequences of this liverwort. Further- more, we show for the first time the specific contribution of identified genes in the liverwort male gametophyte development.

4.2. The potential role of genes specifically expressed in the male thalli of P. endiviifolia sp B

What could be the potential role of proteins encoded by selected genes in the liverwort male gametophyte? We hypothesize the putative involvement of these proteins in the male gametophyte growth and antheridia development as described below.

4.2.1. Genes specifically expressed in the male thalli The expression of PenB_TUA1 and PenB_Raba1/11 genes was observed both in the cultivated in vitro, and grown in the natural habitat, regardless of whether they produced antheridia or not. The lack of expression of both genes in the female gametophytes and Fig. 7. PCR amplicons obtained with genomic DNA template isolated from the female persistent expression of these genes in the male plants indicates their (lane 1) and male (lane 2) thalli. 100 bp ladder is shown in lane 3. involvement in growth and development of male individuals. Earlier 60 I. Sierocka et al. / Gene 485 (2011) 53–62 A

B Fold change Fold change Fold change Fold change

Fig. 8. Analyses of transcript levels of PenB_TUA1, PenB_RABa1/11, PenB_MT and PenB_HMG-box genes using RNA isolated in an independent experiment from: the female thalli without archegonia cultivated in vitro (lane 1), the female thalli producing archegonia collected from the natural habitat (lane 2), the male thalli without antheridia (lane 3) and producing antheridia (lane 4) cultivated in vitro, and the male thalli producing antheridia collected from the natural habitat (lane 5). Specific expression of four identified genes in the male thalli was analyzed by semiquantitative RT-PCR. Negative control is shown in lane 6 and 100 bp ladder in lane 7 (A). Quantitative real-time PCR analyses of four studied genes show different transcripts levels in the male thalli. All transcript levels were normalized against ACTIN1. Calculation shows the mean±SD from three technical replicates (B). cytological studies have shown that the karyotype of P. endiviifolia sp more information about genes specifically expressed during male B is composed of nine chromosomes, in which the smallest ninth gametophyte development. Among them are the genes encoding chromosome has been found invariably to be sexually dimorphic and proteins from tubulin and Rab protein families. Ludwig et al. (1988) usually recognized as an m-chromosome. The ninth chromosome of have found that the TUA1 transcript from A. thaliana accumulates in the male thalli was described as relatively smaller in size, when flowers, but not in leaves or roots of mature plants. The TUA1 compared to the ninth chromosome of the female thalli (Newton, transcript has shown the highest level of expression during anthesis, 1981). The lack of PenB_TUA1 and PenB_Raba1/11 genes in the female suggesting a possible role for TUA1 isotype protein in pollen grain genome may reflect the described differences in the ninth chromo- formation. Silflow's group has found that TUA1 is preferentially some from the male and female gametophyte cells of P. endiviifolia sp expressed in postmitotic pollen grains (Carpenter et al., 1992). Studies B, and may suggest their possible localization on the smaller ninth on α-tubulin gene Tuba1, from non-heading Chinese cabbage chromosome in the plant male cells. The presence of dimorphic sex (Brassica campestris ssp. chinensis) have shown its high level of chromosomes encoding different genes in another liverwort species – expression during all stages of microsporogenesis (Zhang et al., 2009). M. polymorpha (Nakayama et al., 2001) – may suggest that the Yu et al. (2009) reported a pollen-specific TUA protein from Picea presence of sex chromosomes is conserved in dioecious liverworts. It wilsonii. The expression of PwTUA1 is induced by calcium ions and is worth noting that neither of these two genes (nor the two others) boric acid treatments during pollen tube growth, and the over- was found among genes identified in X or Y chromosomes of M. expression of PwTUA1 improved pollen germination and tube growth polymorpha (Yamato et al., 2007). both in P. wilsonii and in A. thaliana. Zhang and coworkers (Yu et al., Whether PenB_TUA1 and PenB_Raba1/11 are involved in the 2009) have suggested that PwTUA1 protein plays a crucial role in development of antheridia is not clear. In seed plants, there is much regulating the deposition of pollen cell wall components in order to I. Sierocka et al. / Gene 485 (2011) 53–62 61 promote pollen germination and tube growth. It seems that there is a deficiency of the essential biotic/abiotic conditions to support proper time-correlation between the expression of genes encoding specific antheridia development, which are ensured only in the natural habitat α-tubulin proteins and the assembling of extensive microtubule of P. endiviifolia sp B. We suggest that PenB_HMG-box and PenB_MT arrays in developing male gametophyte of seed plants. genes are important players in the spermatogenesis process in P. It cannot be excluded that in the liverwort male gametophytes, the endiviifolia sp B. selected PenB_TUA1 gene plays an important role in the development The analysis of physical properties of PenB_HMG-box protein has of antheridia responsible for sperm cells production. Bryophyte sperm shown highly alkaline character of this protein with predicted pI cells are biflagellated motile cells. The spermatogenesis involves 10.72 that is comparable to histone proteins responsible for the development of an elaborate locomotory apparatus of the sperm cell organization of DNA in eukaryotic cells. The HMG-box interacts with that enables its movement and subsequent fertilization of egg cell in the minor groove of DNA with limited or no sequence specificity, the female gametophyte archegonium in the water environment. bending it to different degrees, thus potentially affecting the structure Complex microtubule systems are unique for these cells (Renzaglia et of chromatin. The specific expression of PenB_HMG-box gene in the al., 2000). The presence of specifically expressed α-tubulin transcript male thalli during antheridia development suggests that PenB_HMG- in the male gametophytes of the liverwort P. endiviifolia sp B suggests box protein plays an important role in the organization of the the requirement for the α-tubulin derived from this gene for compacted central nucleus in sperm cells during spermatogenesis in locomotory apparatus assembly of the liverwort sperm cells, as well P. endiviifolia sp B (Renzaglia et al., 2000). The alternative possibility is as for construction of a suitable cytoskeleton for the intracellular the involvement of PenB_HMG-box in the regulation of gene organization of the liverwort male gamete. expression during antheridia development. The HMGB 1, one of the The characteristic organization of seed plants male gametophyte is HMG-box domain-containing proteins from A. thaliana competes with also maintained by a proper regulation of vesicular traffic between H1 histone for binding to DNA. Replacement of this histone by HMGB1 specific cell compartments through endocytotic and exocytotic may induce changes in the chromatin structure, and allows the access pathways. A large family of GTPases, the Rab family proteins, regulates of transcription factors to their target sites in the nucleosome. Another the specificity and vesicle trafficking (Bischoff et al., 1999). Elevated way to promote expression from a given locus by HMG-box proteins is expression of several Rab proteins was found in rapidly growing cells to bend DNA in a manner which results in a spatial conformation or in cells requiring active membrane biogenesis, such as germinating promoting a stable interaction between the promoter site and pollen or flower tissue in Petunia hybrida (PhRAb1), Z. mays (ZmRAB1a, transcription factor, or to recruit successive proteins of the preinitia- ZmRAB1b, ZmRAB1c) and A. thaliana (AtRAB2a)(Jako and Teyssendier tion complex (PIC) (Bianchi and Agresti, 2005; Grasser, 1995). De La Serve, 1996; Palme et al., 1992, 1993). Szumlanski and Nielsen The function of PenB_MT still remains undetermined. No known (2009) have reported that AtRABA4D is specifically expressed in conserved protein domains or motifs were detected in the predicted pollen. Its product was found mainly in the tips of growing pollen PenB_Mt protein with the possible exception of signal peptide and tubes. The loss of function of AtRABA4Dhas led to the disruption of transmembrane domain. Further detailed analyses on structure/ pollen tube polar growth and altered cell wall patterning in vitro, function of this protein are needed. while in vivo, reduced pollen tube guidance at the micropyle was Supplementary materials related to this article can be found online observed. In Nicotiana tabacum overexpression of NtRab11b protein at doi:10.1016/j.gene.2011.06.012. has inhibited pollen tube elongation and affected the directionality of pollen tube growth (de Graaf et al., 2005). Dominant-negative Acknowledgments mutation in the NtRab2 gene inhibited the transport of pollen proteins that enter the secretory pathway and suppressed pollen tube This study was by the Polish Ministry of Higher Education and elongation (Cheung et al., 2002). Together these data indicate the Sciences grant [NN303 815040], [NN303 341635] PhD grant awarded importance of multiple Rab GTPase proteins in male gametophyte to I.S. and the grant for scientific research from the Dean of Biology development and growth in seed plants, most likely due to the Faculty, Adam Mickiewicz University, Poznan, Poland, to I.S. We are regulation of distinct stages of vesicular trafficking in pollen and grateful to Prof. Eva Czarnecka-Verner from the University of Florida regulation of transport of a wide variety of membrane, cell wall, and for the critical review of this manuscript. secretory proteins. The liverwort male gametes, similarly to the pollen tubes of seed References plants, display polarized cellular structure. It is possible that genes encoding specific types of the liverwort Rab proteins, including the Agarwal, P., Reddy, M.K., Sopory, S.K., Agarwal, P.K., 2009. 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