Gene 485 (2011) 53–62 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Novel genes specifically expressed during the development of the male thalli and antheridia in the dioecious liverwort Pellia 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 Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 89 Umultowska Street, 61-614 Poznan, Poland b Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 89 Umultowska Street, 61-614 Poznan, Poland article info abstract Article history: In bryophytes (lower plants), 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. Plant HMG-box proteins bind DNA and may affect chromatin structure, promoting the assembly of nucleoprotein complexes that control DNA-dependent processes including transcription. 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 — Marchantiophyta, class Jungermanniopsida, 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