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Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V β-1,3-glucanase

Abstract

Main conclusion

A gene for β-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination.

Ancestral plant β-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant β-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V β-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

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References

  • Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104–2105

    CAS  Article  PubMed  Google Scholar 

  • Avery GS (1933) Structure and development of the tobacco leaf. Am J Bot 20:565–592

    Article  Google Scholar 

  • Bai F, DeMason DA (2008) Hormone interactions and regulation of PsPK2:GUS compared with DR5:GUS and PID:GUS in Arabidopsis thaliana. Am J Bot 95:133–145

    CAS  Article  PubMed  Google Scholar 

  • Balasubramanian V, Vashisht D, Cletus J, Sakthivel N (2012) Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi. Biotechnol Lett 34:1983–1990

    CAS  Article  PubMed  Google Scholar 

  • Barral P, Suárez C, Batanero E, Alfonso C, Alché JDD, Rodríguez-García MI, Villalba M, Rivas G, Rodríguez R (2005) An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination. Biochem J 390:77–84

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Békésiová I, Nap JP, Mlynárová L (1999) Isolation of high quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Mol Biol Rep 17:269–277

    Article  Google Scholar 

  • Blehová A, Švubová R, Lukačová Z, Moravčíková J, Matušíková I (2015) Transformation of sundew: pitfalls and promises. Plant Cell Tissue Organ Cult 120:681–687

    Article  Google Scholar 

  • Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong X, Agarwal M, Zhu JK (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Gene Dev 17:1043–1054

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • De Smet I, Jürgens G (2007) Patterning the axis in plants—auxin in control. Curr Opin Genet Dev 17:337–343

    Article  PubMed  Google Scholar 

  • Dekkers BJW, Pearce S, van Bolderen-Veldkamp RP, Marshall A, Widera P, Gilbert J, Drost HG, Bassel GW, Müller K, King JR, Wood ATA, Grosse I, Quint M, Krasnogor N, Leubner-Metzger G, Holdsworth MJ, Bentsink L (2013) Transcriptional dynamics of two seed compartments with opposing roles in Arabidopsis seed germination. Plant Physiol 163:205–215

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Doxey AC, Yaish MWF, Moffatt BA, Griffith M, McConkey BJ (2007) Functional divergence in the Arabidopsis β-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states. Mol Biol Evol 24:1045–1055

    CAS  Article  PubMed  Google Scholar 

  • Egan PA, Van Der Kooy F (2013) Phytochemistry of the carnivorous sundew genus Drosera (Droseraceae)—future perspectives and ethnopharmacological relevance. Chem Biodiversity 10:1774–1790

    CAS  Article  Google Scholar 

  • Finch-Savage WE, Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytol 171:501–523

    CAS  Article  PubMed  Google Scholar 

  • Ghanashyam C, Jain M (2009) Role of auxin-responsive genes in biotic stress responses. Plant Signal Behav 4:846–848

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Han X, Hyun T, Zhang M, Kumar R, Koh EJ, Kang BH, Lucas W, Kim JY (2014) Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling. Dev Cell 28:132–146

    CAS  Article  PubMed  Google Scholar 

  • Hatano N, Hamada T (2012) Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata. J Proteomics 75:4844–4852

    CAS  Article  PubMed  Google Scholar 

  • Henrissat B, Davies GJ (2000) Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol 124:1515–1519

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Higo K, Ugawa Y, Iwamoto M, Higo H (1998) PLACE: a database of plant cis-acting regulatory DNA elements. Nucleic Acids Res 26:358–359

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Hirsikorpi M, Kämäräinen T, Teeri T, Hohtola A (2002) Agrobacterium-mediated transformation of round leaved sundew (Drosera rotundifolia L.). Plant Sci 162:537–542

    CAS  Article  Google Scholar 

  • Jain M, Khurana JP (2009) Transcript profiling reveals diverse roles of auxin-responsive genes during reproductive development and abiotic stress in rice. FEBS J 276:3148–3162

    CAS  Article  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jensen MK, Vogt JK, Bressendorff S, Seguin-Orlando A, Petersen M, Sicheritz-Pontén T, Mundy J (2015) Transcriptome and genome size analysis of the Venus flytrap. PLoS One 10(4):e0123887. doi:10.1371/journal.pone.0123887

    Article  PubMed  PubMed Central  Google Scholar 

  • Jongedijk E, Tigelaar H, van Roekel JSC, Bres-Vloemans SA, Dekker I, van den Elzen PJM, Cornelissen BJC, Melchers LS (1995) Synergistic activity of chitinases and β-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 85:173–180

    CAS  Article  Google Scholar 

  • Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649

    Article  PubMed  PubMed Central  Google Scholar 

  • Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371

    CAS  Article  PubMed  Google Scholar 

  • Lenaghan SC, Serpersu K, Xia L, He W, Zhang M (2011) A naturally occurring nanomaterial from the sundew (Drosera) for tissue engineering. Bioinspir Biomim 6:1748–3182

    Article  Google Scholar 

  • Leubner-Metzger G (2002) Seed after-ripening and over-expression of class I β-1,3-glucanase confer maternal effects on tobacco testa rupture and dormancy release. Planta 215:959–968

    CAS  Article  PubMed  Google Scholar 

  • Leubner-Metzger G (2003) Functions and regulation of β-1,3-glucanases during seed germination, dormancy release and after-ripening. Seed Sci Res 13:17–34

    CAS  Article  Google Scholar 

  • Levy A, Erlanger M, Rosenthal M, Epel BL (2007) A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis. Plant J 49:669–682

    CAS  Article  PubMed  Google Scholar 

  • Lyons E, Freeling M (2008) How to usefully compare homologous plant genes and chromosomes as DNA sequences. Plant J 53:661–673

    CAS  Article  PubMed  Google Scholar 

  • Lyons E, Pedersen B, Kane J, Alam M, Ming R, Tang H, Wang X, Bowers J, Paterson A, Lisch D, Freeling M (2008) Finding and comparing syntenic regions among Arabidopsis and the outgroups papaya, poplar, and grape: CoGe with rosids. Plant Physiol 148:1772–1781

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Maddison WP, Maddison DR (2010) Mesquite: a modular system for evolutionary analysis. Mesquite v3.04 (build 725). http://mesquiteproject.org

  • Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, Ke Z, Krylov D, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Thanki N, Yamashita RA, Yin JJ, Zhang D, Bryant SH (2007) CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res 35:D237–D240

    CAS  Article  PubMed  Google Scholar 

  • Matušíková I, Libantová J, Moravčíková J, Mlynárová L, Nap JP (2004) The insectivorous sundew (Drosera rotundifolia, L.) might be a novel source of PR genes for biotechnology. Biologia 59:719–725

    Google Scholar 

  • Meins F, Neuhaus JM, Sperisen C, Ryals J (1992) The primary structure of plant pathogenesis-related glucanohydrolases and their genes. In: Boller T, Meins F (eds) Genes involved in plant defense. Springer, Vienna, pp 245–282

    Chapter  Google Scholar 

  • Michalko J, Socha P, Mészáros P, Blehová A, Libantová J, Moravčíková J, Matušíková I (2013) Glucan-rich diet is digested and taken up by the carnivorous sundew (Drosera rotundifolia L.): implication for a novel role of plant β-1,3-glucanases. Planta 238:715–725

    CAS  Article  PubMed  Google Scholar 

  • Moravčíková J, Matušíková I, Libantová J, Bauer M, Mlynárová L (2004) Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants. Plant Cell Tissue Organ Cult 79:161–168

    Article  Google Scholar 

  • Petersen TN, Brunak S, Von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786

    CAS  Article  PubMed  Google Scholar 

  • Petruzzelli L, Müller K, Hermann K, Leubner-Metzger G (2003) Distinct expression patterns of β-1,3-glucanases and chitinases during the germination of Solanaceous seeds. Seed Sci Res 13:139–153

    CAS  Article  Google Scholar 

  • Piršelová B, Kuna R, Libantová J, Moravčíková J, Matušíková I (2011) Biochemical and physiological comparison of heavy metal-triggered defense responses in the monocot maize and dicot soybean roots. Mol Biol Rep 38:3437–3446

    Article  PubMed  Google Scholar 

  • Piršelová B, Mistríková V, Libantová J, Moravčíková J, Matušíková I (2012) Study on metal-triggered callose deposition in roots of maize and soybean. Biologia 67:698–705

    Google Scholar 

  • Renner T, Specht CD (2012) Molecular and functional evolution of class I chitinases for plant carnivory in the Caryophyllales. Mol Biol Evol 29:2971–2985

    CAS  Article  PubMed  Google Scholar 

  • Rottloff S, Stieber R, Maischak H, Turini FG, Heubl G, Mithoefer A (2011) Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes. J Exp Bot 62:4639–4647

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Schulze WX, Sanggaard KW, Kreuzer I, Knudsen AD, Bemm F, Thogersen IB, Braeutigam A, Thomsen LR, Schliesky S, Dyrlund TF, Escalante-Perez M, Becker D, Schultz J, Karring H, Weber A, Hojrup P, Hedrich R, Enghild JJ (2012) The protein composition of the digestive fluid from the venus flytrap sheds light on prey digestion mechanisms. Mol Cell Proteomics 11:1306–1319

    Article  PubMed  PubMed Central  Google Scholar 

  • Shahmuradov IA, Gammerman AJ, Hancock JM, Bramley PM, Solovyev VV (2003) PlantProm: a database of plant promoter sequences. Nucleic Acids Res 31:114–117

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Sun C, Palmqvist S, Olsson H, Borén M, Ahlandsberg S, Jansson C (2003) A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter. Plant Cell 15:2076–2092

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Teakle GR, Manfield IW, Graham JF, Gilmartin PM (2002) Arabidopsis thaliana GATA factors: organisation, expression and DNA-binding characteristics. Plant Mol Biol 50:43–57

    CAS  Article  PubMed  Google Scholar 

  • Terzaghi W, Cashmore A (1995) Light-regulated transcription. Annu Rev Plant Physiol Plant Mol Biol 46:445–474

    CAS  Article  Google Scholar 

  • Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5:1529–1539

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Urbanowicz BR, Catalá C, Irwin D, Wilson DB, Ripoll DR, Rose JKC (2007) A tomato endo-β-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49). J Biol Chem 282:12066–12074

    CAS  Article  PubMed  Google Scholar 

  • Vatén A, Dettmer J, Wu S, Stierhof YD, Miyashima S, Yadav SR, Roberts CJ, Campilho A, Bulone V, Lichtenberger R, Lehesranta S, Mähönen AP, Kim JY, Jokitalo E, Sauer N, Scheres B, Nakajima K, Carlsbecker A, Gallagher KL, Helariutta Y (2011) Callose biosynthesis regulates symplastic trafficking during root development. Dev Cell 21:1144–1155

    Article  PubMed  Google Scholar 

  • Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An “electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS One 2(8):e718

    Article  PubMed  PubMed Central  Google Scholar 

  • Xie Z, Zhang ZL, Zou X, Huang J, Ruas P, Thompson D, Shen QJ (2005) Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol 137:176–189

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Yachdav G, Kloppmann E, Kajan L, Hecht M, Goldberg T, Hamp T, Hönigschmid P, Schafferhans A, Roos M, Bernhofer M, Richter L, Ashkenazy H, Punta M, Schlessinger A, Bromberg Y, Schneider R, Vriend G, Sander C, Ben-Tal N, Rost B (2014) PredictProtein—an open resource for online prediction of protein structural and functional features. Nucleic Acids Res 42:W337–W343

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Yan D, Duermeyer L, Leoveanu C, Nambara E (2014) The functions of the endosperm during seed germination. Plant Cell Physiol 55:1521–1533

    CAS  Article  PubMed  Google Scholar 

  • Yokoyama R, Nishitani K (2004) Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis. Plant Cell Physiol 45:1111–1121

    CAS  Article  PubMed  Google Scholar 

  • Zur I, Gołebiowska G, Dubas E, Golemiec E, Matušíková I, Libantová J, Moravčíková J (2013) β-1,3-glucanase and chitinase activities in winter triticales during cold hardening and subsequent infection by Microdochium nivale. Biologia 68:241–248

    CAS  Article  Google Scholar 

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Acknowledgments

Financial support was provided by the Operational Programme Research and Development for the project: “Implementation of the research of plant genetic resources and its maintaining in the sustainable management of Slovak republic” (ITMS: 26220220097), co-financed from the resources of the European Union Fund for Regional Development, and by the Slovak Research and Development Agency under the contract no. APVV-15-0051.

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Correspondence to Ildikó Matušíková.

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Michalko, J., Renner, T., Mészáros, P. et al. Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V β-1,3-glucanase. Planta 245, 77–91 (2017). https://doi.org/10.1007/s00425-016-2592-5

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Keywords

  • Cotyledons
  • Embryo hypocotyls
  • Glucanhydrolase
  • PR proteins