, Volume 240, Issue 1, pp 147–159 | Cite as

Structural and functional characteristics of S-like ribonucleases from carnivorous plants

  • Emi Nishimura
  • Shinya Jumyo
  • Naoki Arai
  • Kensuke Kanna
  • Marina Kume
  • Jun-ichi Nishikawa
  • Jun-ichi Tanase
  • Takashi Ohyama
Original Article


Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k cat/K m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k cat/K m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k cat/K m values or k cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.


Droseraceae Enzyme kinetics Nepenthaceae Phylogenetic tree Protein structure Recombinant protein 





Ethylenediaminetetraacetic acid


Isopropyl β-D-thiogalactopyranoside


Polymerase chain reaction





This work was supported by a research grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to T.O.

Supplementary material

425_2014_2072_MOESM1_ESM.pdf (1.3 mb)
Supplementary material 1 (PDF 1281 kb)


  1. Adlassnig W, Peroutka M, Lendl T (2011) Traps of carnivorous pitcher plants as a habitat: composition of the fluid, biodiversity and mutualistic activities. Ann Bot 107:181–194PubMedCentralPubMedCrossRefGoogle Scholar
  2. Anderson B, Midgley JJ (2003) Digestive mutualism, an alternate pathway in plant carnivory. Oikos 102:221–224CrossRefGoogle Scholar
  3. Bariola PA, Green PJ (1997) Plant ribonucleases. In: D’Alessio G, Riordan JF (eds) Ribonucleases: structures and functions. Academic Press, New York, pp 163–190CrossRefGoogle Scholar
  4. Bariola PA, Howard CJ, Taylor CB, Verburg MT, Jaglan VD, Green PJ (1994) The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J 6:673–685PubMedCrossRefGoogle Scholar
  5. Bodenhausen N, Reymond P (2007) Signaling pathways controlling induced resistance to insect herbivores in Arabidopsis. Mol Plant Microbe Interact 20:1406–1420PubMedCrossRefGoogle Scholar
  6. Corbishley TP, Johnson PJ, Williams R (1984) Serum ribonuclease. In: Hans UB, Jürgen B, Marianne G (eds) Methods of enzymatic analysis, vol 4. Verlag Chemie, Weinheim, pp 134–143Google Scholar
  7. Deshpande RA, Shankar V (2002) Ribonucleases from T2 family. Crit Rev Microbiol 28:79–122PubMedCrossRefGoogle Scholar
  8. Dlakic M, Harrington RE (1998) DIAMOD: display and modeling of DNA bending. Bioinformatics 14:326–331PubMedCrossRefGoogle Scholar
  9. Dodds PN, Clarke AE, Newbigin E (1996) Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation. Plant Mol Biol 31:227–238PubMedCrossRefGoogle Scholar
  10. Ellison AM, Gotelli NJ (2009) Energetics and the evolution of carnivorous plants—Darwin’s ‘most wonderful plants in the world’. J Exp Bot 60:19–42PubMedCrossRefGoogle Scholar
  11. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, New York, pp 571–607CrossRefGoogle Scholar
  12. Hayashi T, Kobayashi D, Kariu T, Tahara M, Hada K, Kouzuma Y, Kimura M (2003) Genomic cloning of ribonucleases in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection, and characterization of their promoters. Biosci Biotechnol Biochem 67:2574–2583PubMedCrossRefGoogle Scholar
  13. Hillwig MS, Contento AL, Meyer A, Ebany D, Bassham DC, Maclntosh GC (2011) RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants. Proc Natl Acad Sci USA 108:1093–1098PubMedCentralPubMedCrossRefGoogle Scholar
  14. Hino M, Kawano S, Kimura M (2002) Expression of Nicotiana glutinosa ribonucleases in Escherichia coli. Biosci Biotechnol Biochem 66:910–912PubMedCrossRefGoogle Scholar
  15. Igic B, Kohn J (2001) Evolutionary relationships among self-incompatibility RNases. Proc Natl Acad Sci USA 98:13167–13171PubMedCentralPubMedCrossRefGoogle Scholar
  16. Jost W, Bak H, Glund K, Terpstra P, Beintema JJ (1991) Amino acid sequence of an extracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur J Biochem 198:1–6PubMedCrossRefGoogle Scholar
  17. Kamiya H, Fukunaga S, Ohyama T, Harashima H (2007) The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo. Arch Biochem Biophys 461:7–12PubMedCrossRefGoogle Scholar
  18. Kao T, Huang S (1994) Gametophytic self-incompatibility: a mechanism for self/nonself discrimination during sexual reproduction. Plant Physiol 105:461–466PubMedCentralPubMedGoogle Scholar
  19. Kariu T, Sano K, Shimokawa H, Itoh R, Yamasaki N, Kimura M (1998) Isolation and characterization of a wound-inducible ribonuclease from Nicotiana glutinosa leaves. Biosci Biotechnol Biochem 62:1144–1151PubMedCrossRefGoogle Scholar
  20. Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371PubMedCrossRefGoogle Scholar
  21. Köck M, Löffler A, Abel S, Glund K (1995) cDNA structure and regulatory properties of a family of starvation- induced ribonucleases from tomato. Plant Mol Biol 27:477–485PubMedCrossRefGoogle Scholar
  22. Köck M, Groß N, Stenzel I, Hause G (2004) Phloem-specific expression of the wound-inducible ribonuclease LE from tomato (Lycopersicon esculentum cv. Lukullus). Planta 219:233–242PubMedCrossRefGoogle Scholar
  23. LeBrasseur ND, MacIntosh GC, Pérez-Amador MA, Saitoh M, Green PJ (2002) Local and systemic wound-induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA-independent systemic signaling pathway. Plant J 29:393–403PubMedCrossRefGoogle Scholar
  24. Lers A, Khalchitski A, Lomaniec E, Burd S, Green PJ (1998) Senescence-induced RNases in tomato. Plant Mol Biol 36:439–449PubMedCrossRefGoogle Scholar
  25. Liu Y, Cotton JA, Shen B, Han X, Rossiter SJ, Zhang S (2010) Convergent sequence evolution between echolocating bats and dolphins. Curr Biol 20:R53–R54PubMedCrossRefGoogle Scholar
  26. Löffler A, Abel S, Jost W, Beintema JJ, Glund K (1992) Phosphate-regulated induction of intracellular ribonucleases in cultured tomato (Lycopersicon esculentum) cells. Plant Physiol 98:1472–1478PubMedCentralPubMedCrossRefGoogle Scholar
  27. Löffler A, Glund K, Irie M (1993) Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. FEBS J 214:627–633CrossRefGoogle Scholar
  28. Luhtala N, Parker R (2010) T2 family ribonucleases: ancient enzymes with diverse roles. Trends Biochem Sci 35:253–259PubMedCentralPubMedCrossRefGoogle Scholar
  29. Ma RC, Oliveira MM (2000) The RNase PD2 gene of almond (Prunus dulcis) represents an evolutionarily distinct class of S-like RNase genes. Mol Gen Genet 263:925–933PubMedCrossRefGoogle Scholar
  30. MacIntosh GC, Hillwig MS, Meyer A, Flagel L (2010) RNase T2 genes from rice and the evolution of secretory ribonucleases in plants. Mol Genet Genomics 283:381–396PubMedCrossRefGoogle Scholar
  31. Mccubbin AG, Kao T (2000) Molecular recognition and response in pollen and pistil interactions. Annu Rev Cell Dev Biol 16:333–364PubMedCrossRefGoogle Scholar
  32. Nishimura E, Kawahara M, Kodaira R, Kume M, Arai N, Nishikawa J, Ohyama T (2013) S-like ribonuclease gene expression in carnivorous plants. Planta 238:955–967PubMedCrossRefGoogle Scholar
  33. Okabe T, Iwakiri Y, Mori H, Ogawa T, Ohyama T (2005a) An S-like ribonuclease gene is used to generate a trap-leaf enzyme in the carnivorous plant Drosera adelae. FEBS Lett 579:5729–5733PubMedCrossRefGoogle Scholar
  34. Okabe T, Futatsuya C, Tanaka O, Ohyama T (2005b) Structural analysis of the gene encoding Drosera adelae S-like ribonuclease DA-I. J Adv Sci 17:218–224CrossRefGoogle Scholar
  35. Peroutka M, Adlassnig W, Lendl T, Pranic K, Lichtscheidl IK (2008) Functional biology of carnivorous plants. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant biotechnology: Advances and topical issues. Global Science Books, Isleworth, pp 266–287Google Scholar
  36. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786PubMedCrossRefGoogle Scholar
  37. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612PubMedCrossRefGoogle Scholar
  38. Richards JH (2001) Bladder function in Utricularia purpurea (Lentibulariaceae): is carnivory important? Am J Bot 88:170–176PubMedCrossRefGoogle Scholar
  39. Rojas HJ, Roldán JA, Goldraij A (2013) NnSR1, a class III non-S-RNase constitutively expressed in styles, is induced in roots and stems under phosphate deficiency in Nicotiana alata. Ann Bot 112:1351–1360PubMedCrossRefGoogle Scholar
  40. Sato K, Egami F (1957) Studies on ribonucleases in Takadiastase. I. J Biochem 44:753–767Google Scholar
  41. Shimizu T, Inoue T, Shiraishi H (2001) A senescence-associated S-like RNase in the multicellular green alga Volvox carteri. Gene 274:227–235PubMedCrossRefGoogle Scholar
  42. Stevens PF (2001 onwards). Angiosperm phylogeny website. Version 13, July 2012. Accessed 13 Feb 2014
  43. Sumida N, Nishikawa J, Kishi H, Amano M, Furuya T, Sonobe H, Ohyama T (2006) A designed curved DNA segment that is a remarkable activator of eukaryotic transcription. FEBS J 273:5691–5702PubMedCrossRefGoogle Scholar
  44. Takahashi K, Matsumoto K, Nishii W, Muramatsu M, Kubota K (2009) Comparative studies on the acid proteinase activities in the digestive fluids of Nepenthes, Cephalotus, Dionaea, and Drosera. Carniv Plant Newsl 38:75–82Google Scholar
  45. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCentralPubMedCrossRefGoogle Scholar
  46. Tanaka N, Arai J, Inokuchi N, Koyama T, Ohgi K, Irie M, Nakamura KT (2000) Crystal structure of a plant ribonuclease, RNase LE. J Mol Biol 298:859–873PubMedCrossRefGoogle Scholar
  47. Taylor CB, Bariola PA, delCardayré SB, Raines RT, Green PJ (1993) RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc Natl Acad Sci USA 90:5118–5122PubMedCentralPubMedCrossRefGoogle Scholar
  48. Theologis A, Ecker JR, Palm CJ, Federspiel NA, Kaul S et al (2000) Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana. Nature 408:816–820PubMedCrossRefGoogle Scholar
  49. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604PubMedCrossRefGoogle Scholar
  50. Van Nerum I, Certal AC, Oliveira MM, Keulemans J, Broothaerts W (2000) PD1, an S-like RNase gene from a self-incompatible cultivar of almond. Plant Cell Rep 19:1108–1114CrossRefGoogle Scholar
  51. Wilson CM (1975) Plant nucleases. Annu Rev Plant Physiol 26:187–208CrossRefGoogle Scholar
  52. Ye ZH, Droste DL (1996) Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans. Plant Mol Biol 30:697–709PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Emi Nishimura
    • 1
  • Shinya Jumyo
    • 1
  • Naoki Arai
    • 1
  • Kensuke Kanna
    • 1
  • Marina Kume
    • 1
  • Jun-ichi Nishikawa
    • 2
  • Jun-ichi Tanase
    • 2
  • Takashi Ohyama
    • 1
    • 2
  1. 1.Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and EngineeringWaseda UniversityTokyoJapan
  2. 2.Department of Biology, Faculty of Education and Integrated Arts and SciencesWaseda UniversityTokyoJapan

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