Unravel- ing the molecular mechanisms of apple flowering would open the way for ... (Malus x domestica SOC1, MdSOC1), which is a member of the relatively.
Communications
in
Agricultural
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Applied Biological Sciences
Volume 71(1) 1-330 (2006) – Communications in Agricultural and Applied Biological Sciences
Vol 71(1) 1-330 (2006)
Vol 71(1) 1-330 (2006)
COMMUNICATIONS IN AGRICULTURAL AND APPLIED BIOLOGICAL SCIENCES formerly known as MEDEDELINGEN FACULTEIT LANDBOUWKUNDIGE EN TOEGEPASTE BIOLOGISCHE WETENSCHAPPEN
PUBLISHERS Professors
Pascal Boeckx Peter Bossier Guy Smagghe Walter Steurbaut Els Van Damme Erick Vandamme Niko Verhoest
Editorial address Coupure links 653 9000 Gent (Belgium) Website
http://www.fbw.ugent.be ISSN 1379-1176
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ISOLATION AND CHARACTERIZATION OF A MADS-BOX TYPE GENE FROM APPLE (MALUS X DOMESTICA)
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N. MAHNA1,2,3, R. DREESEN1, B. BAGHBAN KOHNEH ROUZ2, B. GHAREYAZIE3, M. VALIZADEH2, V. GRIGORIAN2 & J. KEULEMANS1 Lab for Fruit Breeding and Biotechnology, Center for fruit Culture (R&D Division, KULeuven), W. de Croylaan 42, Heverlee, BE-3001, Belgium 2 Department of Agronomy and Plant Breeding, Faculty of Agriculture University of Tabriz, 51664, Tabriz, Iran 3 Department of Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII) Seed and Plant Improvement Institutes Campus P.O. Box 31535-1897, Mahdasht Road, Karaj, Iran
INTRODUCTION Apple tree (Malus x domestica) has an extended juvenile phase of several years, during which vegetative growth is maintained. This characteristic is recognized as a disadvantage in breeding and stable annual production. Therefore it is necessary to gain an understanding of the genetic mechanisms underlying transition from vegetative to reproductive phase. Unraveling the molecular mechanisms of apple flowering would open the way for understanding and manipulating juvenility, which in turn can be applied in other fruit trees as well, especially those belonging to the Rosaceae family. Different flowering genes have already been identified and characterized in apple. Some are as shown in Table1. Table 1. A brief summary of flowering genes already isolated and characterized from apple. Apple gene MdMADS1 MdMADS2 MdMADS3 MdMADS4 MdMADS5 MdMADS10 MdCOL1 and MdCOL2 MdPI MdTFL
Arabidopsis homologue SEP SQUAMOSA SEP SEP AP1 AG CO PI TFL1
Reference Sung and An, 1997 Sung et al. 1999 Sung et al. 2000 Sung et al. 2000 Yao et al., 1999 Yao et al., 1999 Jeong et al., 1999 Yao et al., 2001 Kotoda et al., 2003
In this paper, we report the discovery of an additional MADS-box type gene (Malus x domestica SOC1, MdSOC1), which is a member of the relatively poorly characterized SOC1/TM3 class of MADS-box genes. MATERIALS AND METHODS Apple EST sequence data were downloaded from the Genome Database for Rosaceae (http://www.mainlab.clemson.edu/gdr/) and the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/). Apple ESTs similar to the SOC1 gene (GenBank accession numbers: CV998019; CV880272; CV657904; CO722859) were identified and assem-
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bled to make a contig. An ORF translation was made which showed a strong homology with the SOC1 protein sequence. Translation and protein alignments were performed with ClustalX (Thompson et al., 1994) and other software. Primers (sense: 5’-AAGAGAGTGGGCATCCACAA-3’; antisense: 5’GAATGTTGACTTTAGACTGC-3’) designed from the MdSOC1 contig so that the amplified fragment would include the ORF. Young leaves of apple trees growing in the greenhouse were collected, quickly frozen in liquid nitrogen and stored at -80°C until further use. RNA was extracted according to the LiCl-protocol (Sambrook et al., 1989). In this protocol a DNase-treatment is included to eliminate possible contaminating DNA in preparation for the Reverse Transcription (RT) reactions. The reverse transcription reaction was carried out in 20 µl reaction volume following the protocol supplied with SUPERSCRIPT II reverse transcriptase kit (Invitrogen). The resulting cDNAs were used directly as templates for PCR. PCR conditions were as follows: 2’ 94°C, followed by 32 cycles of 94°C (30”), 55°C (30”) and 72°C (1’), ending with final extension at 72°C for 10’ and using Platinum® Taq DNA Polymerase (Invitrogen). The resulting fragment was cloned into the pGEM® -T Easy vector (Promega, Madison, WI) and was transformed into DH5α-type competent cells. The fragment sequenced using the M13 universal primers. The resulting sequence data for the coding region have been deposited in the GenBank database under accession number DQ846833. RESULTS AND DISCUSSION MADS-box proteins are transcription factors that control a diverse range of developmental processes in plants (Becker and Theissen, 2003). They are characterized by a highly conserved N-terminal DNA-binding domain termed the “MADS-box”. The MADS-box gene family contains more than 100 members in Arabidopsis and comprises of five major clades, of which only one, the so-called “MIKC” class, has been the subject of significant functional analysis (Becker and Theissen, 2003). SOC1 is a member of this “MIKC class” that integrates flowering signals from the photoperiod, vernalization, and gibberellin pathways (Borner et al.2000; Lee et al., 2000) (Figure 1).
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Figure 1. Arabidopsis genes regulating flowering time and development (Lemmetyinen, 2003)
Therefore it could be of importance to be able to manipulate the expression of this gene to change the time of flowering and growth habit. Here, we report the isolation of a Malus x domestica homolog of the SOC1 (MdSOC1) gene. The resulting sequence contains a part of 5’ UTR, a coding sequence and a part of 3’ UTR. BLAST homology searches of the coding region of this gene revealed a high homology with the SOC1 protein from Arabidopsis. The coding region contains a K-box named so because of its structural similarity to the coiled-coil domain of Keratin (Ma et al. 1991, Theissen et al. 1995). The K-box domain mediates protein-protein interactions of the MADS-box proteins to form homo- or heterodimers facilitating DNA-binding to other regulatory factors (Ma et al. 1991, Pnueli et al. 1991, Theissen et al. 1995). Furthermore, a conserved domain of SOC1, the so-called “SOC1 motif” (Nakamura et al. 2005, Vandenbussche et al. 2003) has been identified in the C-terminal region. The similarity between the MADS-box of MdSOC1 assembled contig and SOC1 protein from Arabidopsis is 85.7% while that of K-box is 71.8% and C-terminal SOC1 motif is 75%.The resulting sequence had a gap at 5’ of the coding region comparing to the contig, and consequently, it had just one quarter of MADS box (Figure 3). This gene is probably a 5’-truncated form of the complete gene which could be the result of an alternative splicing event as a way to regulate functionality. Expression analysis of this gene showed that it is expressed in young leaves but not in the terminal buds (Figure 2).
Figure 2. Expression pattern of MdSOC1 in terminal buds and young leaves. Actin was used as an internal control.
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More than one-fifth of plant genes are estimated to be alternatively spliced (Wang and Brendel, 2006). However, how to distinguish erroneous splicing from functional events is yet to be resolved. The coding region of the isolated (possibly exposed to alternative splicing) MdSOC1 has still an ATG start codon in its ORF and could be translated as a protein containing K-box and C-terminal motif. Nevertheless, its functionality is not known and it could be concluded that this probable alternative splicing event could exert a special functional role for silencing of the gene or for assigning a new function to it. Further experiments are yet to be carried out to isolate the complete gene as well as to elucidate its possible function.
Figure 3. Alignment of the amino acid sequence of the isolated MdSOC1 against the assembled contig of EST sequences and the SOC1 protein of Arabidopsis. As it is clear, there is a high homology between these sequences. A region at the 5’ terminus of the coding sequence, which is a part of the MADS-box consensus, is lacking as the result of an alternative splicing event.
ACKNOWLEDGEMENTS The authors wish to thank the Iranian Ministry of Science, Research and technology for granting a visiting scholarship to N.M.
REFERENCES BECKER A 1 THEISSEN G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phyl Evol 29:464-489. BORNER R, KAMPMANN G, CHANDLER J, GLEISSNER R, WISMAN E, APEL K & MELZER S (2000) A MADS domain gene involved in the transition to flowering in Arabidopsis. Plant J 24:591–599. JEONG DH, SUNG SK & AN G (1999) Molecular Cloning and Characterization of CONSTANS-Like cDNA Clones of the Fuji Apple. Journal of Plant Biology 42(1):23-31.
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KOTODA N, WADA M, MASUDA T & SOEJIMA J (2003) The break-through in the reduction of juvenile phase in apple using transgenic approaches. Acta Horticulturae 625:337-343. LEE H, SUH S, PARK E, CHO E, AHN JH, KIM S, LEE JS, KWON YM & LEE I (2000) The AGAMOUS-LIKE20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev 14:2366–2376. LEMMETYINEN J (2003) The birch genes BpMADS1 and BpMADS6 and their use in the modification of flowering. PhD Dissertation in Biology, University of Joensuu, 85 pp. MA H, YANOFSKY MF & MEYEROWITZ EM (1991) AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev 5:484495. MOON J, SUH SS, LEE H, CHOI KR, HONG CB, PAEK NC, KIM SG & LEE I (2003) The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. Plant J 35:613–623. NAKAMURA T, SONG I, FUKUDA T, YOKOYAMA J, MAKI M, OCHIAI T, KAMEYA T & KANNO A (2005) Characterization of TrcMADS1 gene of Trillium camtschatcense (Trilliaceae) reveals functional evolution of the SOC1/TM3-like gene family. J Plant Res. 118(3): 229-234. PNUELI L, ABU-ABEID M, ZAMIR D, NACKEN W, SCHWARZ-SOMMER Z & LIFSCHITZ E (1991) The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes from Antirrhinum and Arabidopsis. Plant J 1(2):255–266. SAMBROOK J, FRITSCH EF & MANIATIS T (1989) Molecular Cloning, a Laboratory Manual. 2nd Edition, Cold Spring Harbor, Cold Spring Harbor Laboratory Press SUNG SK & AN G (1997) Molecular cloning and characterization of a MADS-box cDNA clone of the Fuji apple. Plant Cell Physiol 38(4):484-9. SUNG SK, YU GH & AN G (1999) Characterization of MdMADS2, a Member of the SQUAMOSA Subfamily of Genes, in Apple. Plant Physiol. 120:969-978. SUNG SK, YU GH, NAM J, JEONG DH & AN G (2000) Developmentally regulated expression of two MADS-box genes, MdMADS3 and MdMADS4, in the morphogenesis of flower buds and fruits in apple. Planta 210(4):519-528. THEISSEN G, STRATER T, FISCHER A & SAEDLER H (1995) Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUSlike MADS-box genes from maize. Gene 156:155-166 THOMPSON JD, HIGGINS DG & GIBSON TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspecific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. VANDENBUSSCHE M, THEISSEN G, VAN DE PEER Y & GERATS T (2003) Structural diversification and neo-functionalization during floral MADS-box gene evolution by Cterminal frameshift mutations. Nucleic Acids Res 31:4401–4409. WANG BB & BRENDEL V (2006) Genomewide comparative analysis of alternative splicing in plants. PNAS 103:7175-7180. YAO JL, DONG YH, KARNHEDEN A & MORRIS B (1999) Seven MADS-box genes in apple are expressed in different parts of the fruit. J Amer Hort Sci 124:8-13. YAO J, DONG Y & MORRIS BA (2001) Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. PNAS 98(3):1306-11. Abbreviations: AG, AGAMOUS; AP1, APETALA1; CO, CONSTANS; EST, Expressed Sequence Tag; MdMADS1, Malus domestica MADS 1; PI, PISTILLATA; MdPI, Malus domestica PI; PTM5, Populus tremuloides MADS-box 5; RT, Reverse Transcription; SEP, SEPALLATA; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CO 1; TM3, TOMATO MADS 3; Malus domestica TFL (MdTFL); TFL1, TERMINAL FLOWER 1.
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