InSilico Analysis for Five Major Cereal Crops Phytocystatins

  • Rupal ChauhanEmail author
  • Yogesh Jasrai
  • Himanshu Pandya
Original Research Article


Five major cereal crops such as rice, wheat, maize, barley and sorghum are continuously threatened by a multitude of pathogens and other disorders. Cystatins offers a pivotal role in deciding the promising plant response. The use of bioinformatics tools for phylogenetic relationships of five major cereal crop (rice, wheat, maize, barley and sorghum) phytocystatins based on amino acid sequence information was elucidated, and their secondary and tertiary structures were investigated for structural comparisons. Twenty-eight distinct phytocystatins from 28 plant species were investigated. Phytocystatins could be divided into five distinct phylogenetic groups. Five major cereal crops their structural features were highly conserved, and their amino acid sequence similarities ranged from 48 to 86 %. A new highly conserved amino acid sequence motif, YEAKxWxKxF, in the C-terminal end being unique to phytocystatins was identified. The predicted 3D homology models showed a high conservation of the general central structure of the phytocystatins, i.e., the 4–5 anti-parallel \(\beta\)-sheets, wrapping halfway round a single central \(\alpha\)-helix and particularly the three active site regions, the N-terminal, the first and second hairpin loops. Any structural differences seem to be mainly in the length of the N- and C-terminal, the length of the second hairpin loop and the fifth \(\beta\)-sheet. Via docking experiments, small heterogeneities were observed in the vicinity of the OC-I active sites that seemed to be influential in the binding process and stability of the resultant inhibitor–protease complex.


Five major cereal crop phytocystatin Phylogenetic relationship Structural comparisons 


  1. 1.
    Shriparna D, Singh VK, Soma SM, Anil K (2010) In silico analysis of sequential, structural and functional diversity of wheat cystatins and its implication in plant defense. Genomic Proteomics Bioinform 8(1):42–56CrossRefGoogle Scholar
  2. 2.
    Turk V, Bode W (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285(2):213–219CrossRefGoogle Scholar
  3. 3.
    Ryan SN, Liang WA, McManus MT (1989) A cysteine proteinase inhibitor purified from apple fruit. Phytochemistry 49:957–963CrossRefGoogle Scholar
  4. 4.
    Haas BJ, Volfovsky N, Town CD, Troukhan M, Alexandrov N, Feldmann KA, Flavell RB, White O, Salzberg SL (2002) Full-length messenger RNA sequences greatly improve genome annotation. Genome Biol 3(6):1465–6906CrossRefGoogle Scholar
  5. 5.
    Gaddour K, Vicente-Carbajosa J, Lara P, Isabel-Lamoneda I, Diaz I, Carbonero P (2001) A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli. Plant Mol Biol 45:599–608CrossRefGoogle Scholar
  6. 6.
    Lim CO, Lee SI, Chung WS, Park SH, Hwang I, Cho MJ (1996) Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds. Plant Mol Biol 30(2):373–379CrossRefGoogle Scholar
  7. 7.
    Sugawara H, Shibuya K, Yoshioka T, Hashiba T, Satoh S (2002) Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers? J Exp Bot 53(368):407–413CrossRefGoogle Scholar
  8. 8.
    Szederkenyi J, Komor E, Schobert C (1995) cDNA expressed in Ricinus cotyledons. Plant Physiol 109:721–722CrossRefGoogle Scholar
  9. 9.
    Pernas M, Sanchez-Monge R, Gomez L, Salcedo G (1998) A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests. Plant Mol. Biol. 38(6):1235–1242CrossRefGoogle Scholar
  10. 10.
    Gholizadeh A, Santha IM, Kohnehrouz BB, Lodha ML, Kapoor HC (2005) Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock’s comb). Appl Biochem 42(3):197–204CrossRefGoogle Scholar
  11. 11.
    Abe M, Abe K, Kuroda M, Arai S (1992) Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Molecular cloning and expression studies. Eur J Biochem 209(3):933–937CrossRefGoogle Scholar
  12. 12.
    Fernandes KV, Sabelli PA, Barratt DH, Richardson M, Xavier-Filho J, Shewry PR (1993) The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors. Plant Mol Biol 23(1):215–219CrossRefGoogle Scholar
  13. 13.
    Rassam M, Laing WA (2004) Purification and characterization of phytocystatins from kiwifruit cortex and seeds. Phytochemistry 65(1):19–30CrossRefGoogle Scholar
  14. 14.
    Hubinger G, Gruber P, Vollmann U, Kraft D, Ferreira F, Himly M (1999) Molecular cloning and nucleotide sequence analysis of a cDNA (Accession No. AF143677) encoding the cystatin homolog from Mugwort Artemisia vulgaris) Pollen. Plant Physiol 120(2):634Google Scholar
  15. 15.
    Abe K, Emori Y, Kondo H, Suzuki K, Arai S (1987) Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds. J Biol Chem 262(35):16793–16797PubMedGoogle Scholar
  16. 16.
    Song I, Taylor M, Baker K, Bateman RC Jr (1995) Inhibition of cysteine proteases by Carica papaya cystatin produced in Escherichia coli. Gene 162(2):221–224CrossRefGoogle Scholar
  17. 17.
    Walsh TA, Strickland JA (1993) Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains. Plant Physiol 103(4):1227–1234CrossRefGoogle Scholar
  18. 18.
    Li Z, Sommer A, Dingermann T, Noe CR (1996) Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor from Sorghum bicolor seedlings. Mol Gen Genet 251(4):499–502PubMedGoogle Scholar
  19. 19.
    Zhao Y, Botella MA, Subramanian L, Niu X, Nielsen SS, Bressan RA, Hasegawa PM (1996) Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol 111(4):1299–1306CrossRefGoogle Scholar
  20. 20.
    Botella MA, Xu Y, Prabha TN, Zhao Y, Narasimhan ML, Wilson KA, Nielsen SS, Bressan RA, Hasegawa PM (1996) Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl assonate. Plant Physiol 112(3):1201–1210CrossRefGoogle Scholar
  21. 21.
    To KY, Suen DF, Chen SCG (1999) A sweet potato leaf cDNA (Accession No. AF117334) encoding cysteine proteinase inhibitor. Plant Physiol 119(4):1568Google Scholar
  22. 22.
    Huang YJ, To KY, Yap MN, Chiang WJ, Suen DF, Chen SC (2001) Cloning and characterization of leaf senescence up-regulated genes in sweet potato. Physiol Plant. 113(3):384–391CrossRefGoogle Scholar
  23. 23.
    Yang AH, Yeh KW (2005) Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no.1). Planta 221:493–501CrossRefGoogle Scholar
  24. 24.
    Girarad C, Rivard D, Kiggundu A, Kunert K, Gleddie SC, Cloutier C, Michaud D (2007) A multicomponent, elicitor-inducible cystatin complex in tomato, Solanum lycopersicum. New Phytol 173(4):841–851CrossRefGoogle Scholar
  25. 25.
    Kuroda M, Kiyosaki T, Matsumoto I, Misaka T, Arai S, Abe K (2001) Molecular cloning, characterization, and expression of wheat cystatins. Biosci Biotechnol Biochem 65(1):22–28CrossRefGoogle Scholar
  26. 26.
    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. Nucleic Acids Res 25:4876–4882CrossRefGoogle Scholar
  27. 27.
    Felsenstein J (1989) PHYLIP-phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  28. 28.
    Page RDM (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12(4):357–358PubMedGoogle Scholar
  29. 29.
    Koichiro T, Daniel P, Nicholas P, Glen S, Masatoshi N, Sudhir K (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739CrossRefGoogle Scholar
  30. 30.
    Nagata K, Kudo N, Abe K, Arai S, Tanokura M (2000) Three-dimensional solution of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica. Biochemistry 39:14753–14760CrossRefGoogle Scholar
  31. 31.
    Stubbs MT, Laber B, Bode W, Huber R, Jerala R, Lenarcic B, Turk V (1990) The refined 2.4 Å X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interactionD. EMBO J 9:1939–1947CrossRefGoogle Scholar
  32. 32.
    Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissing H, Shindyalov IN, Bourne PE (2000) The protein data bank. nucleic Acids Res 28:235–242CrossRefGoogle Scholar
  33. 33.
    Ambrish R, Alper K, Yang Z (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Nat Protoc 5:725–738CrossRefGoogle Scholar
  34. 34.
    Laskowski RA, MacArthur MW, Moss DS, Thomton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291CrossRefGoogle Scholar
  35. 35.
    Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdfViewer. An environment for comparative protein modeling. Electrophoresis 18:2714–2723CrossRefGoogle Scholar
  36. 36.
    Margis R, Reis EM, Villeret V (1998) Structural and phylogenetic relationships among plant and animal cystatins. Arch Biochem Biophys 359:24–30CrossRefGoogle Scholar
  37. 37.
    Martinez M, Diaz Z (2008) The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship. BMC Evol Biol 8:198CrossRefGoogle Scholar

Copyright information

© International Association of Scientists in the Interdisciplinary Areas and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Applied Botany Center, Department of Botany, University School of SciencesGujarat UniversityAhmadabadIndia

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