Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume–rhizobia symbiosis


Root nodule (RN) symbiosis is a mutualistic interaction observed between nitrogen-fixing soil bacteria and nodulating plants, which are scattered in only four orders of angiosperms called nitrogen-fixing clade. Most of legumes engage in RN symbiosis with rhizobia. Molecular genetic analyses with legumes and non-leguminous nodulating plants revealed that RN symbiosis utilizes early signalling components that are required for symbiosis with arbuscular mycorrhizal (AM) fungi. However detailed evolutionary processes are still largely unknown. Comparative analyses with non-nodulating species phylogenetically related to legumes could be better strategies to study the evolution of RN symbiosis in legumes. Polygala paniculata is a non-leguminous species that belongs to a family different from legumes but that is classified into the same order, Fabales. It has appropriate characteristics for cultivation in laboratories: small body size, high fertility and short lifecycles. Therefore, we further assessed whether this species is suitable as a model species for comparative studies with legumes. We first validated that the plant we obtained in Palau was truly P. paniculata by molecular phylogenetic analysis using rbcL sequences. The estimated genome size of this species was less than those of two model legumes, Lotus japonicus and Medicago truncatula. We determined conditions for cultivation in vitro and for hairy root formation from P. paniculata seedlings. It would facilitate to investigate gene functions in this species. The ability of P. paniculata to interact with AM fungi was confirmed by inoculation with Rhizophagus irregularis, suggesting the presence of early signalling factors that might be involved in RN symbiosis. Unexpectedly, branching of root hairs was observed when inoculated with Mesorhizobium loti broad host range strain NZP2037, indicating that P. paniculata has the biological potential to respond to rhizobia. We propose that P. paniculata is used as a model plant for the evolutionary study of RN symbiosis.

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  1. Andrew SM, Moe SR, Totland Ø et al (2012) Species composition and functional structure of herbaceous vegetation in a tropical wetland system. Biodivers Conserv 21:2865–2885

  2. Aoki S, Syono K (1999) Synergistic function of rolB, rolC, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in hairy root induction in Nicotiana tabacum. Plant Cell Physiol 40:252–256

  3. Banks H, Klitgaard BB, Claxton F et al (2008) Pollen morphology of the family Polygalaceae (Fabales). Bot J Linn Soc 156:253–289

  4. Bello MA, Bruneau A, Forest F et al (2009) Elusive relationships within order Fabales: phylogenetic analyses using matK and rbcL sequence data. Syst Bot 34:102–114

  5. Bello MA, Rudall PJ, Hawins JA (2012) Combined phylogenetic analyses reveal interfamilial relationships and patterns of floral evolution in the eudicot order Fabales. Cladistics 28:393–421

  6. Bonfante P, Genre A (2010) Mechanisms underlying beneficial plant–fungus interactions in mycorrhizal symbiosis. Nat Commun 1:48

  7. Broughton WJ, Dilworth MJ (1971) Control of leghemoglobin synthesis in Snake beans. Biochem J 125:1075–1080

  8. Brundrett M (2009) Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil 320:37–77

  9. Castro S, Loureiro J, Rodriguez E et al (2007) Evaluation of polysomaty and estimation of genome size in Polygala vayredae and P. calcarea using flow cytometry. Plant Sci 172:1131–1137

  10. CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sci USA 106:12794–12797

  11. Choi J, Summers W, Paszkowski U (2018) Mechanisms underlying establishment of arbuscular mycorrhizal symbioses. Annu Rev Phytopathol 56:135–160

  12. Christenhusz MJM, Byng JW (2016) The number of known plants species in the world and its annual increase. Pytotaxa 261:201–217

  13. Coelho VPM, Agra MF, Baracho GS (2008) Flora da Paraíba, Brasil: Polygala L. (Polygalaceae). Acta Bot Bras 22:225–239

  14. de Faria SM, Lewis GP, Sprent JI et al (1989) Occurrence of nodulation in the Leguminosae. New Phytol 111:607–619

  15. Doyle JJ (2011) Phylogenetic perspectives on the origins of nodulation. Mol Plant Microbe Int 24:1289–1295

  16. Favarger C, Huynh KL (1965) Polygala paniculata L., 2n = 52–56. In Á. Löve (ed) IOPB IOPB chromosome number reports IV. Taxon 14:86–92

  17. Fay MF, Bayer C, Alverson WS et al (1998) Plastid rbcL sequence data indicate a close affinity between Diegodendron and Bixa. Taxon 47:43–50

  18. Forest F, Chase MW, Persson C et al (2007) The role of biotic and abiotic factors in evolution of ant dispersal in the milkwort family (Polygalaceae). Evolution 61:1675–1694

  19. Frescura VD, Laughinghouse HD 4th, do Canto-Dorow TS et al (2012) Pollen viability of Polygala paniculata L. (Polygalaceae) using different staining methods. Biocell 36:143–145

  20. Godwin I, Todd G, Ford-Lloyd B et al (1991) The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep 9:671–675

  21. Griesmann M, Chang Y, Liu X et al (2018) Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science 361:1743

  22. Handa Y, Nishide H, Takeda N et al (2015) RNA-seq transcriptional profiling of an arbuscular mycorrhiza provides insights into regulated and coordinated gene expression in Lotus japonicus and Rhizophagus irregularis. Plant Cell Physiol 56:1490–1511

  23. Huynh KL (1965) Contribution à l’étude caryologique et embryologique des phanérogames du Pérou. Denkschriften der schweizerischen naturforschenden Gesellschaft 85:1–178

  24. Imaizumi-Anraku H, Takeda N, Charpentier M et al (2005) Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. Nature 433:527–531

  25. Ito M, Miyamoto J, Mori Y et al (2000) Genome and chromosome dimensions of Lotus japonicus. J Plant Res 113:435–442

  26. Johann S, Mendes BG, Missau FC et al (2011) Antifungal activity of five species of Polygala. Braz J Microbiol 42:1065–1075

  27. Kasai-Maita H, Hirakawa H, Nakamura Y et al (2013) Commonalities and differences among symbiosis islands of three Mesorhizobium loti strains. Microbes Environ 28:275–278

  28. Käss E, Wink M (1996) Molecular evolution of the Fabaceae: phylogeny of the three subfamilies based on rbcL-sequences. Biochem Syst Ecol 24:365–378

  29. Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20:1160–1166.

  30. Kelly S, Sullivan J, Ronson C et al (2014) Genome sequence of the Lotus spp. microsymbiont Mesorhizobium loti strain NZP2037. Stand Genom Sci 9:7

  31. Kistner C, Parniske M (2002) Evolution of signal transduction in intracellular symbiosis. Trends Plant Sci 7:511–518

  32. Kouchi H, Imaizumi-Anraku H, Hayashi M et al (2010) How many Peas in a Pod? Legume genes responsible for mutualistic symbioses underground. Plant Cell Physiol 51:1381–1397

  33. Kumar V, Sharma A, Narasimha Prasad BC et al (2006) Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Electron J Biotechnol 9:4

  34. Lack AJ (1995) Relationships and hybridization between British species of Polygala evidence from isozymes. New Phytol 130:217–223

  35. Lewis WH, Davis SA (1962) Cytological observations of Polygala in eastern North America. Rhodora 64:102–113

  36. Madsen LH, Tirichine L, Jurkiewicz A et al (2010) The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus. Nat Commun 1:10

  37. Mennes CB, Moerland MS, Rath M et al (2015) Evolution of mycoheterotrophy in Polygalaceae: the case of Epirixanthes. Am J Bot 102:598–608

  38. Miwa H, Sun J, Oldroyd GE et al (2006) Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus. Mol Plant Microbe Int 19:914–923

  39. NCBI Resource Coordinators (2016) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 44 (D1):D7–D19. Accessed 30 March 2017.

  40. Nogueira FLP, Fernandes SBO, Reis GM et al (2005) Atividade analgésica e antiedematogênica de Polygala paniculata L. (Polygalaceae) selvagem e obtida por micropropagação. Braz J Pharmacogn 15:310–315

  41. Okamoto S, Yoro E, Suzaki T et al (2013) Hairy root transformation in Lotus japonicus. Bio Protoc 3:e795

  42. Oldrody GE (2013) Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11:252–263

  43. Paiva JAR (1998) Polygalarum africanarum et madagascariensium prodromus atque gerontogaei generis Heterosamara Kuntze, a genere Polygala L. segregati et a nobis denuo recepti, synopsis monographica. Madrid: Cyanus. Fontqueria 50:1–346

  44. Pankhurst CE, Hopcroft DH, Jones WT (1987) Comparative morphology and flavolan content of Rhizobium loti induced effective and ineffective root nodules on Lotus species, Leuceana leucocephala, Carmichaelia flagelliformis, Ornithopus sativus, and Clianthus puniceus. Can J Bot 65:2676–2685

  45. Parniske M (2008) Arbuscular mycorrhiza: the mother of plant root endosymbiosis. Nat Rev Microbiol 6:763–775

  46. Qiu YL, Li L, Wang B et al (2010) Angiosperm phylogeny inferred from sequences of four mitochondrial genes. J Syst Evol 48:391–425

  47. R Core Team (2016) R: A language and environment for statistical computing. Accessed 30 March 2017.

  48. Rath M, Weber HC, Imhof S (2013) Morpho-anatomical and molecular characterization of the mycorrhizas of European Polygala species. Plant Biol 15:548–557

  49. Rath M, Grolig F, Haueisen J et al (2014) Combining microtomy and confocal laser scanning microscopy for structural analyses of plant-fungus associations. Mycorrhiza 24:293–300

  50. Remy W, Taylor TN, Hass H et al (1994) Four hundred-million-year-old vesicular arbuscular mycorrhizae. Proc Natl Acad Sci USA 91:11841–11843

  51. Royal Botanic Gardens Kew (2017) Seed information database (SID). Version 7.1. Accessed 30 March 2017.

  52. Saito K, Yoshikawa M, Yano K et al (2007) NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus. Plant Cell 19:610–624

  53. Sato S, Nakamura Y, Kaneko T et al (2008) Genome structure of the legume, Lotus japonicus. DNA Res 15:227–239

  54. Sharma ML, Mehra PN (1978) Chromosome numbers in some north west Indian species of Polygala. Cytologia 43:589–593

  55. Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298

  56. Simon L, Bousquet J, Lévesque RC et al (1993) Origin and diversification of endomycorrhizal fungi and coincidence with vascular land plants. Nature 363:67–69

  57. Smit P, Limpens E, Geurts R et al (2007) Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling. Plant Physiol 145:183–191

  58. Soltis PS, Soltis DE, Chase MW (1999) Angiosperm phylogeny inferred from multiple genes as a tool for comparative biology. Nature 402:402–404

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

  60. Stevens PF (2001) Angiosperm Phylogeny Website. Version 14. Accessed 29 May 2019.

  61. Stubblefield SP, Taylor TN, Trappe JM (1987) Fossil mycorrhizae: a case for symbiosis. Science 237:59–60

  62. Sulaiman SF, Culham A, Harborne JB (2003) Molecular phylogeny of Fabaceae based on rbcL sequence data: with special emphasis on the tribe Mimoseae (Mimosoideae). Asia Pac J Mol Biol Biotechnol 11:9–35

  63. Suyama M, Torrents D, Bork P (2006) PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res 34:W609–W612

  64. Takeda N, Tsuzuki S, Suzaki T et al (2013) CERBERUS and NSP1 of Lotus japonicus are common symbiosis genes that modulate arbuscular mycorrhiza development. Plant Cell Physiol 54:1711–1723

  65. Takeda N, Handa Y, Tsuzuki S et al (2015) Gibberellins interfere with symbiosis signaling and gene expression and alter colonization by arbuscular mycorrhizal fungi in Lotus japonicus. Plant Physiol 167:545–557

  66. Tang H, Krishnakumar V, Bidwell S et al (2014) An improved genome release (version Mt4.0) for the model legume Medicago truncatula. BMC Genom 15:312

  67. The Angiosperm Phylogeny Group (2016) An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Bot J Linn Soc 181:1–20

  68. van Velzen R, Holmer R, Bu F et al (2018) Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses. Proc Natl Acad Sci USA 115:E4700–E4709

  69. van Velzen R, Doyle JJ, Geurts R (2019) Resurrected scenario: single gain and massive loss of nitrogen-fixing nodulation. Trends Plant Sci 24:49–57

  70. Werner GDA, Cornwell WK, Sprent JI et al (2014) A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms. Nat Commun 5:4087

  71. Yang TYA, Chen CF (2013) A revision of the genus Polygala L. (Polygalaceae) in Taiwan. Taiwania 58:156–162

  72. Yano K, Aoki S, Liu M et al (2017) Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads. DNA Res 24:193–203

  73. Young ND, Debellé F, Oldroyd GE et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520–524

  74. Zhu H, Riely BK, Burns NJ et al (2006) Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics 172:2491–2499

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The seed samples were permitted to export by Palau Government with the certificate (No. ROP-018-2014). Authors thank Ms. Sachiko Tanaka (NIBB) for preliminary experiments and kind support on germination condition, seedling growth and seed harvesting. We thank Mr. Naoki Morooka and model plant research facility at NIBB for the analyses of genome size using CyFlow SL. This study was financially supported by Grant-in-Aid for Scientific Research on Innovative Areas (16H01248 to HF and 16H06279 to WI) of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and Grant-in-Aid for Scientific Research (C) (16K08149 to TS and 17K07509 to SA) of Japan Society for the Promotion of Science.

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Tokumoto, Y., Hashimoto, K., Soyano, T. et al. Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume–rhizobia symbiosis. J Plant Res 133, 109–122 (2020).

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  • Fabales
  • Genome size
  • Hairy root
  • Polygala paniculata
  • rbcL
  • RN symbiosis
  • Root hair response