Genome-wide identification and molecular characterization of cysteine protease genes in rice

  • Marjohn C. Niño
  • Me-Sun Kim
  • Kwon Kyoo KangEmail author
  • Yong-Gu ChoEmail author
Original Article


Cysteine protease activity comprises the majority of proteolytic activities in plants. They are involved in almost every facet of the plant’s development. Accumulating evidence indicates multiple roles of this protease type in response to biotic and abiotic stress. To understand the regulations and functions of cysteine protease in rice, its evolutionary and structural evidence was uncovered in this study. Using MEROPS, a peptidase database, the 74 rice cysteine proteases belonging to six families were queried. Each of these families represents distinct proteolytic enzyme; C1 is a papain-like protease, C2 is a calpain-2-type, C12 is an ubiquitinyl hydrolase-L1 enzyme, C13 is legumain, C14 is a caspase-1 type, and C15 is a pyroglutamyl peptidase 1 enzyme type. Evolutionary expansion attributed to gene duplication and diversification was particularly evident in C1 family which showed the highest number (n = 53) of members, most of which contained the highest number and most variable introns and motifs, whereas families C13, C14, and C15 had only a few members which all contained lesser number and variation of intron and motif. Out of 74 total cysteine protease gene members, 73 were globular proteins and 55 were predicted as stable proteins. Spatial expression assay of selected C1 members showed that LOC_Os01g73980 and LOC_Os05g01810 were highly expressed in the stem and leaves, while LOC_Os02g27030 was constitutively expressed in all tissues. The expression of LOC_Os01g73980 and LOC_Os05g01810 was also highly activated by salinity stress, while LOC_Os02g27030 was activated by both salinity and heat. LOC_Os05g01810 overexpression transgenic rice exhibited moderate tolerance to salinity stress, which provides interesting clues on biological functions of these genes in rice.


Cysteine protease Papain-like protease Salinity stress Rice 



This research was supported by the Next-Generation BioGreen 21 Program (The Agricultural Genome Center, No. PJ01330201), Rural Development Administration, and by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Golden Seed Project funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (213009-05-3-WT211), Republic of Korea.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

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Supplementary material 1 (TIFF 1970 kb)
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Supplementary material 2 (TIFF 625 kb)


  1. Amit M, Donyo M, Hollander D, Goren A, Kim E, Gelfman S, Lev-Maor G, Burstein D, Schwartz S, Postolsky B, Pupko T, Ast G (2012) Differential GC content between exons and introns establishes distinct strategies of splice-site recognition. Cell Rep 1(5):543–556PubMedCrossRefGoogle Scholar
  2. Atsushi I (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895–1898Google Scholar
  3. Bailey TL, Elkan C (1994) Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2:28–36PubMedPubMedCentralGoogle Scholar
  4. Beynon R, Bond JS (2000) Proteolytic enzymes, 2nd edn. Series Practical Approach. Oxford University Press, OxfordGoogle Scholar
  5. Carels N, Bernardi G (2000) Two classes of genes in plants. Genetics 154(4):1819–1825PubMedPubMedCentralGoogle Scholar
  6. Chen JM, Fortunato M, Barrett AJ (2000) Activation of human prolegumain by cleavage at a C-terminal asparagine residue. Biochem J 352:327–334PubMedPubMedCentralCrossRefGoogle Scholar
  7. Chen JM, Dando PM, Barrett AJ (2004) Mammalian legumain. In: Barrett AJ, Rawlings ND, Woessner JF (eds) Handbook of proteolytic enzymes, 2nd edn. Elsevier, London, pp 1302–1305Google Scholar
  8. Choudhuri S (2014) Bioinformatics for beginners: genes, genomes, molecular evolution, databases and analytical tools. Academic Press, New York. ISBN 978-0-12-410471-6Google Scholar
  9. Clement Y, Fustier M-A, Nabholz B, Glemin S (2015) The bimodal distribution of genic GC content is ancestral to monocot species. Genome Biol Evol 7(1):336–348CrossRefGoogle Scholar
  10. Dando PM, Fortunato M, Strand GB, Smith TS, Barrett AJ (2003) Pyroglutamyl-peptidase I: cloning, sequencing, and characterization of the recombinant human enzyme. Protein Expr Purif 28:111–119PubMedCrossRefGoogle Scholar
  11. Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68:383–424PubMedCrossRefGoogle Scholar
  12. Goodall GJ, Filipowicz W (1989) The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58(3):473–483PubMedCrossRefGoogle Scholar
  13. Goodall GJ, Filipowicz W (1991) Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants. EMBO J 10(9):2635–2644PubMedPubMedCentralCrossRefGoogle Scholar
  14. Grudkowska M, Zagdanska B (2004) Multifunctional role of plant cysteine proteinases. Acta Biochim Pol 51:609–624PubMedGoogle Scholar
  15. Hayashi Y, Yamada K, Shimada T, Matsushima R, Nishizawa NK, Nishimura M, Hara-Nishimura I (2001) A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiol 42:894–899PubMedCrossRefGoogle Scholar
  16. Hemelaar J, Borodovsky A, Kessler BM, Reverter D, CookJ Kolli N, Gan-Erdene T, Wilkinson KD, Gill G, Lima CD, Ploegh HL, Ovaa H (2004) Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol Cell Biol 24:84–95PubMedPubMedCentralCrossRefGoogle Scholar
  17. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300PubMedPubMedCentralCrossRefGoogle Scholar
  18. Jain M, Khurana P, Tyagi AK, Khurana JP (2008) Genome-wide analysis of intronless genes in rice and Arabidopsis. Funct Integr Genom 8:69–78CrossRefGoogle Scholar
  19. Johnston SC, Riddle SM, Cohen RE, Hill CP (1999) Structural basis for the specificity of ubiquitin C-terminal hydrolases. EMBO J 18:3877–3887PubMedPubMedCentralCrossRefGoogle Scholar
  20. Jones JT, Mullet JE (1995) A salat and dehydration-inducible pea gene, Cyp15a, encodes a cell-wall protein with sequence similarity to cysteine protease. Plant Mol Biol 28:1055–1065PubMedCrossRefPubMedCentralGoogle Scholar
  21. Karrer KM, Peiffer SL, Ditomas ME (1993) Two distinct gene subfamilies within the family of cysteine protease genes. Proc Natl Acad Sci USA 90:3063–3067PubMedCrossRefGoogle Scholar
  22. Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, Shinozaki K (1993) Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene 129:175–182PubMedCrossRefGoogle Scholar
  23. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874PubMedPubMedCentralCrossRefGoogle Scholar
  24. Liu Z, Chen S, Xie L, Lu Z, Liu M, Han X, Qiao G, Jiang J, Zhuo R, Qiu Q, He Z (2017) Overexpression of cysteine protease gene from Salix matsudana enhances salt tolerance in transgenic Arabidopsis. Environ Exp Bot 147:53–62Google Scholar
  25. Lodish H, Berk A, Zipursky SL (2000) Molecular cell biology, 4th edn. W. H. Freeman, New YorkGoogle Scholar
  26. Lu Y, Song Z, Lu K, Lian X, Cai H (2012) Molecular characterization, expression and functional analysis of the amino acid transporter gene family (OsAATs) in rice. Acta Physiol Plant 34:1943–1962CrossRefGoogle Scholar
  27. Niño MC, Kim JK, Lee HJ, Abdula SE, Nou IS, Cho YG (2014) Key roles of cysteine protease in different plant pathosystem. Plant Breed Biotech 2(2):97–109CrossRefGoogle Scholar
  28. Niño MC, Nogoy FM, Kang KK, Cho YG (2018) Low-affinity cation transporter 1 improves salt stress tolerance in Japonica rice. Plant Breed Biotech 6(1):82–93CrossRefGoogle Scholar
  29. Parsell DA, Sauer RT (1989) The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. J Biol Chem 264(13):7590–7595PubMedGoogle Scholar
  30. Rawlings ND, Morton FR, Barrett AJ (2006) MEROPS: the peptidase database. Nucl Acids Res 34:270–272CrossRefGoogle Scholar
  31. Rawlings ND, Barrett AJ, Bateman A (2010) MEROPS: the peptidase database. Nucl Acids Res 38:227–233CrossRefGoogle Scholar
  32. Rawlings ND, Waller M, Barrett AJ, Bateman A (2014) MEROPS: the database of proteolytic enzymes, their substrates, and inhibitors. Nucl Acids Res 42:503–509CrossRefGoogle Scholar
  33. Ressayre A, Glemin S, Montalent P, Serre-Giardi L, Dillmann C, Joets J (2015) Introns structure patterns of variation in nucleotide composition in Arabidospsis thaliana and rice protein-coding genes. Genome Biol Evol 7(10):2913–2928PubMedPubMedCentralCrossRefGoogle Scholar
  34. RStudio Team (2015) RStudio: integrated development for R. RStudio, Inc., Boston, MA,
  35. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425Google Scholar
  36. Sato K, Saito Y, Kawashima S (1995) Identification and characterization of membrane-bound calpains in clathrin-coated vesicles from bovine brain Eur. J Biochem 230:25–31Google Scholar
  37. Shabalina SA, Spiridonov NA, Kashina A (2013) Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity. Nucl Acids Res 41(4):2073–2094PubMedCrossRefGoogle Scholar
  38. Shindo T, van der Hoorn RAL (2008) Papain-like cysteine proteases: key players at molecular battlefields employed by both plants and their invaders. Mol Plant Pathol 9:119–125PubMedGoogle Scholar
  39. Sorimachi H (2004) Mu-Calpain. In: Barrett AJ, Rawlings ND, Woessner JF (eds) Handbook of Proteolytic Enzymes, 2nd edn. Elsevier, London, pp 1206–1211Google Scholar
  40. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  41. Travaglini-Allocatelli C, Ivarsson Y, Jemth P, Gianni S (2009) Folding and stability of globular proteins and implications for function. Curr Opin Struct Biol 19(1):3–7PubMedCrossRefGoogle Scholar
  42. van der Hoorn RAL, Leeuwenburgh MA, Bogyo M, Joosten MHAJ, Peck SC (2004) Activity profiling of papain-like cysteine proteases in plants. Plant Physiol 135:1170–1178PubMedPubMedCentralCrossRefGoogle Scholar
  43. Wang C, Barry JK, Min Z, Tordsen G, Rao AG, Olsen OA (2003) The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase. J Biol Chem 278:34467–34474PubMedCrossRefGoogle Scholar
  44. Wang D, Pei K, Fu Y, Sun Z, Li S, Liu H, Tang K, Han B, Tao Y (2007) Genome-wide analysis of the auxin response factors ARF gene family in rice Oryza sativa. Gene 394:13–24PubMedCrossRefGoogle Scholar
  45. Warnecke T, Weber CC, Hurst LD (2009) Why there is more to protein evolution than protein function: splicing, nucleosomes and dual-coding sequence. Biochem Soc Trans 37:756–761PubMedCrossRefGoogle Scholar
  46. Weatheritt RJ, Babu MM (2013) The hidden codes that shape protein evolution. Science 342(6164):1325–1326PubMedPubMedCentralCrossRefGoogle Scholar
  47. Wilkinson KD, Laleli-Sahin E, Urbauer J, Larsen CN, Shih GH, Haas AL, Walsh ST, Wand AJ (1999) The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3. J Mol Biol 291:1067–1077PubMedCrossRefGoogle Scholar
  48. Yamaguchi-Shinozaki K, Koizumi M, Urao S, Shinozaki K (1992) Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol 33:217–224CrossRefGoogle Scholar

Copyright information

© Korean Society for Plant Biotechnology 2019

Authors and Affiliations

  1. 1.Department of Crop ScienceChungbuk National UniversityCheongjuKorea
  2. 2.Center for Studies in BiotechnologyCebu Technological University Barili CampusCagay, BariliPhilippines
  3. 3.Department of HorticultureHankyong National UniversityAnseongKorea

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