title: The activity of TcCYS4 modified by variations in pH and temperature can affect symptoms of witches' broom disease of cocoa, caused by the fungus Moniliophthora perniciosa.
abstract: The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches' broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches' broom disease in cocoa.
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brazil.
authors.forename: Ana Camila Oliveira
authors.initials: AC
authors.lastname: Freitas
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brazil.
authors.forename: Cristiane Ferreira
authors.initials: CF
authors.lastname: Souza
authors.affiliation: Centro de Biologia e Ciêncais da Saúde, Universidade do Norte do Paraná, Londrina, Paraná, Brazil.
authors.forename: Paulo Sérgio
authors.initials: PS
authors.lastname: Monzani
authors.affiliation: Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), Santo André, Sao Paulo, Brazil.
authors.forename: Wanius
authors.initials: W
authors.lastname: Garcia
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brazil.
authors.forename: Alex Alan Furtado
authors.initials: AA
authors.lastname: de Almeida
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brazil.
authors.forename: Marcio Gilberto Cardoso
authors.initials: MG
authors.lastname: Costa
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brazil.
authors.forename: Carlos Priminho
authors.initials: CP
authors.lastname: Pirovani
mesh_terms: D000363:Agaricales / Q000502:physiology*; D002099:Cacao / Q000378:metabolism / Q000382:microbiology*; D015891:Cystatins / Q000378:metabolism*; D060467:Disease Resistance; D006863:Hydrogen-Ion Concentration; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000378:metabolism / Q000382:microbiology; D010940:Plant Proteins / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D058767:Protein Unfolding; D044366:Transition Temperature
chemical_list: D015891:Cystatins; D010940:Plant Proteins
references.citation: Plant Mol Biol. 2008 Sep;68(1-2):131-43
references.pmid: 18523728
references.citation: Proc Natl Acad Sci U S A. 1989 Apr;86(7):2214-8
references.pmid: 16594020
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Plant Cell. 1999 Mar;11(3):431-44
references.pmid: 10072402
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Electrophoresis. 2008 Jun;29(11):2391-401
references.pmid: 18435495
references.citation: J Exp Bot. 2005 Mar;56(413):865-77
references.pmid: 15642708
references.citation: FEBS J. 2013 Feb;280(4):1028-38
references.pmid: 23241243
references.citation: FEMS Microbiol Lett. 2007 Sep;274(2):238-44
references.pmid: 17610511
references.citation: Planta. 2010 Nov;232(6):1485-97
references.pmid: 20859638
references.citation: Curr Opin Plant Biol. 2004 Aug;7(4):400-7
references.pmid: 15231262
references.citation: Nat Struct Biol. 2001 Apr;8(4):316-20
references.pmid: 11276250
references.citation: Insect Biochem Mol Biol. 2002 Apr;32(4):405-15
references.pmid: 11886775
references.citation: Mycologia. 2005 Sep-Oct;97(5):1012-22
references.pmid: 16596953
references.citation: FEBS Lett. 2007 Jun 26;581(16):2914-8
references.pmid: 17543305
references.citation: Mol Plant Pathol. 2008 Sep;9(5):577-88
references.pmid: 19018989
references.citation: J Mol Biol. 2005 Sep 9;352(1):202-11
references.pmid: 16061250
references.citation: Plant Physiol Biochem. 2013 Dec;73:254-65
references.pmid: 24161755
references.citation: Mol Cell Biol. 1993 Dec;13(12):7652-65
references.pmid: 8246982
references.citation: Physiol Plant. 2014 Jan;150(1):1-17
references.pmid: 23586401
references.citation: Arch Biochem Biophys. 1998 Nov 1;359(1):24-30
references.pmid: 9799556
references.citation: Amyloid. 2001 Mar;8(1):1-10
references.pmid: 11293820
references.citation: J Mol Biol. 2004 Feb 6;336(1):165-78
references.pmid: 14741212
references.citation: Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1416-20
references.pmid: 8108423
references.citation: Acta Biochim Pol. 2004;51(3):609-24
references.pmid: 15448724
references.citation: BMC Genomics. 2008 Nov 18;9:548
references.pmid: 19019209
references.citation: J Agric Food Chem. 2007 Mar 7;55(5):1762-6
references.pmid: 17295505
references.citation: Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3127-31
references.pmid: 8159715
references.citation: J Biomol Struct Dyn. 2013;31(6):649-64
references.pmid: 22881286
references.citation: J Biol Chem. 1996 Jan 19;271(3):1314-21
references.pmid: 8576118
references.citation: Mol Plant Microbe Interact. 2008 Jul;21(7):891-908
references.pmid: 18533830
references.citation: Annu Rev Phytopathol. 1996;34:573-94
references.pmid: 15012557
references.citation: Exp Cell Res. 2004 Jul 15;297(2):607-18
references.pmid: 15212960
references.citation: Ann Bot. 2007 Jul;100(1):129-40
references.pmid: 17557832
references.citation: Plant Physiol Biochem. 2011 Aug;49(8):917-22
references.pmid: 21641227