Abstract
Phytocystatins encompass a family of plant competitive cysteine proteinase inhibitors. They are encoded by part of a conserved monophyletic group of genes that are found in all eukaryotes. The primary targets of phytocystatins are papain-like cysteine proteinases. However, a group of larger phytocystatins is also able to inhibit proteinases such as legumains. Phytocystatins have been implicated in several physiological processes and act within an intricate proteolytic regulatory network. The present work characterizes the gene family of rice phytocystatins, which contains twelve genes with different features. Phylogenetic analyses cluster rice phytocystatins into three main groups. Group 1 is composed of OcI, OcIII and OcXII and is nearly ubiquitous and highly expressed in plants under normal and stressed conditions including salt, wounding, ABA or a fungal elicitor such as chitosan. Rice phytocystatins can contribute to plant senescence and may exhibit an inverse correlation between their gene expression and the activities of their target proteinases. This work contributes to clarifying the roles of individual phytocystatin genes in plant processes such as germination and response to environmental stresses.
Similar content being viewed by others
References
Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104–2105
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. J Biol Chem 262:16793–16797
Agrawal GK, Rakwal R, Tamogami S, Yonekura M, Kubo A, Saji H (2002) Chitosan activates defense/stress response(s) in the leaves of Oryza sativa seedlings. Plant Physiol Biochem 40:1061–1069
Alvarez-Fernandez M, Barrett a J, Gerhartz B, Dando PM, Ni J, Abrahamson M (1999) Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site. J Biol Chem 274:19195–19203
Arai S, Watanabe H, Kondo H (1991) Papain activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members. J Biochem 109:294–298
Arai S, Matsumoto I, Abe K (1998) Phytocystatins and their target enzymes: from molecular biology to practical application: a review. J Food Biochem 22:287–299
Arai S, Matsumoto I, Emori Y, Abe K (2002) Plant seed cystatins and their target enzymes of endogenous and exogenous origin. J Agric Food Chem 50:6612–6617
Arenhart RA, Bai Y, Oliveira LFV, Neto LB, Schunemann M, Maraschin F, Mariath J, Silverio A, Sachetto-Martins G, Margis R, Wang Z-Y, Margis-Piheiro M (2013) New insights into aluminum tolerance in rice: the ASR5 protein binds the STAR1 promoter and other aluminum-responsive genes. Mol Plant 55:1–14
Beers EP, Woffenden BJ, Zhao C (2000) Plant proteolytic enzymes: possible roles during programmed cell death. Plant Mol Biol 44:399–415
Belenghi B, Acconcia F, Trovato M, Perazzolli M, Bocedi A, Polticelli F, Ascenzi P, Delledone M (2003) AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. Eur J Biochem 270:2593–2604
Benchabane M, Schlüter U, Vorster J, Goulet M-C, Michaud D (2010) Plant cystatins. Biochimie 92:1657–1666
Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V (1988) The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J 7:2593
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cao P, Jung K-H, Choi D, Hwang D, Zhu J, Ronald PC (2012) The rice oligonucleotide array database: an atlas of rice gene expression. Rice 5:17
Chan Y-L, Yang A-H, Chen J-T, Yeh K-W, Chan M-T (2010) Heterologous expression of taro cystatin protects transgenic tomato against Meloidogyne incognita infection by means of interfering sex determination and suppressing gall formation. Plant Cell Rep 29:231–238
Christoff AP, Turchetto-Zolet AC, Margis R (2014) Uncovering legumain genes in rice. Plant Sci 215–216:100–109
Chu M-H, Liu K-L, Wu H-Y, Yeh K-W, Cheng Y-S (2011) Crystal structure of tarocystatin-papain complex: implications for the inhibition property of group-2 phytocystatins. Planta 234:243–254
Davidson RM, Gowda M, Moghe G, Lin H, Vaillancourt B, Shiu SH, Jiang N, Robin Buell C (2012) Comparative transcriptomics of three Poaceae species reveals patterns of gene expression evolution. Plant J 71:492–502
Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29:1969–1973
Dutt S, Singh VK, Marla SS, Kumar A (2010) In silico analysis of sequential, structural and functional diversity of wheat cystatins and its implication in plant defense. Genomics, proteomics Bioinforma/Beijing Genomics Inst 8:42–56
Francis SE, Ersoy RA, Ahn J-W, Atwell BJ, Roberts TH (2012) Serpins in rice: protein sequence analysis, phylogeny and gene expression during development. BMC Genom 13:449
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellstern U, Puntnam N, Rokshar DS (2011) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 22:1–9
Goulet M-C, Dallaire C, Vaillancourt L-P, Khalf M, Badri AM, Preradov A, Duceppe M-O, Goulet C, Cloutier C, Michaud D (2008) Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites. Plant Physiol 146:1010–1019
Grudkowska M, Zagdańska B (2004) Multifunctional role of plant cysteine proteinases. Acta Biochim Polonica 51:609–624
Guo K, Bu Y, Takano T, Liu S, Zhang X (2013) Arabidopsis cysteine proteinase inhibitor AtCYSb interacts with a Ca(2+)-dependent nuclease, AtCaN2. FEBS Lett 587:3417–3421
Hara-Nishimura I, Hatsugai N (2011) The role of vacuole in plant cell death. Cell Death Differ 18:1298–1304
Hara-Nishimura I, Inoue K, Nishimura M (1991) A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett 294:89–93
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 42:894–899
Hong JK, Hwang JE, Lim CJ, Yang KA, Jin Z-L, Kim CY, Koo JC, Chung WS, Lee KO, Lee SY, Cho MJ, Lim CO (2007) Over-expression of Chinese cabbage phytocystatin 1 retards seed germination in Arabidopsis. Plant Sci 172:556–563
Hwang JE, Hong JK, Je JH, Lee KO, Kim DY, Lee SY, Lim CO (2009) Regulation of seed germination and seedling growth by an Arabidopsis phytocystatin isoform, AtCYS6. Plant Cell Rep 28:1623–1632
Kawahara Y, Oono Y, Kanamori H, Matsumoto T, Itoh T, Minami E (2012) Simultaneous RNA-seq analysis of a mixed transriptome of rice and blast fungus interaction. PLoS One 7:e49423
Khanna-Chopra R, Srivalli B, Ahlawat YS (1999) Drought induces many forms of cysteine proteases not observed during natural senescence. Biochem Biophys Res Commun 255:324–327
Kondo H, Abe K, Nishimura I, Watanabe H, Emori Y, Arai S (1990) Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. J Biol Chem 265:15832–15837
Lalitha S, Shade RE, Murdock LL, Bressan RA, Hasegawa PM, Nielsen SS (2005) Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests. Comp Biochem Physiol C: Toxicol Pharmacol 140:227–235
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Lee SC, Luan S (2012) ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant, Cell Environ 35:53–60
Lepelley M, Amor Ben M, Martineau N, Cheminade G, Caillet V, McCarthy J (2012) Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination. BMC Plant Biol 12:31
Letunic I, Doerks T, Bork P (2012) SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res 40:D302–D305
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408
Margis R, Reis EM, Villeret V (1998) Structural and phylogenetic relationships among plant and animal cystatins. Arch Biochem Biophys 359:24–30
Margis-Pinheiro M, Zolet ACT, Loss G, Pasquali G, Margis R (2008) Molecular evolution and diversification of plant cysteine proteinase inhibitors: new insights after the poplar genome. Mol Phylogenet Evol 49:349–355
Martinez M, Diaz I (2008) The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship. BMC Evol Biol 8:198
Martinez M, Diaz-Mendoza M, Carrillo L, Diaz I (2007) Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases. FEBS Lett 581:2914–2918
Martinez M, Cambra I, Carrillo L, Diaz-Mendonza M, Diaz I (2009) Characterization of the entire cystatin gene family in barley and their target cathepsin L-like cysteine-proteases, partners in the hordein mobilization during seed germination. Plant Physiol 151:1531–1545
Martínez M, Abraham Z, Carbonero P, Díaz I (2005) Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley. Mol Genet Genomics 273:423–432
Martínez M, Cambra I, González-Melendi P, Santamaría ME, Díaz I (2012) C1A cysteine-proteases and their inhibitors in plants. Physiol Plant 145:85–94
Milne I, Stephen G, Bayer M, Cock PJ, Pritchard L, Cardle L, Shaw PD, Marshall D (2013) Using Tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193–202
Novinec M, Lenarčič B (2013) Papain-like peptidases: structure, function, and evolution. BioMol Concepts 4:287–308
Ohtsubo S, Kobayashi H, Noro W, Taniguchi M, Saitoh E (2005) Molecular cloning and characterization of oryzacystatin-III, a novel member of phytocystatin in rice (Oryza sativa L. japonica). J Agric Food Chem 53:5218–5224
Park S-O, Yu J-W, Park J-S, Li J, Yoo S-C, Lee N-Y, Lee S-K, Jeong S-W, Seo HS, Koh H-J, Jeon J-S, Park Y-I, Paek N-C (2007) The senescence induced staygreen protein regulates chlorophyll degradation. Plant Cell 19:1649–1664
Pierre O, Hopkins J, Combier M, Baldacci F, Engler G, Brouguisse R, Hérouart D, Boncompagni E (2014) Involvement of papain and legumain proteinase in the senescence process of Medicago truncatula nodules. New Phytol 202:849–863
Pirovani CP, da Silva Santiago A, dos Santos LS, Micheli F, Margis R, da Silva Gesteira A, Alvim FC, Pereira GA, de Mattos Cascardo JC (2010) Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms. Planta 232:1485–1497
Quain MD, Makgopa ME, Márquez-García B, Comadira G, Fernandez-Garcia N, Olmos E, Schnaubelt D, Kunert KJ, Foyer CH (2014) Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits. Plant Biotechnol J 1–11. doi:10.1111/pbi.12193
Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116–W120
Rambaldi D, Ciccarelli FD (2009) FancyGene: dynamic visualization of gene structures and protein domain architectures on genomic loci. Bioinformatics 25:2281–2282
Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJ, Moorman AF (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37:e45
Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–444
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 28:2731–2739
Thompson J, Higgins Gibson T (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Turk V, Bode W (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285:213–219
Valdes-Rodriguez S, Cedro-Tanda A, Aguilar-Hernandez V, Cortes-Onofre E, Blanco-Labra A, Guerrero-Rangel A (2010) Recombinant amaranth cystatin (AhCPI) inhibits the growth of phytopathogenic fungi. Plant Physiol Biochem 48:469–475
van der Hoorn RA, Jones JD (2004) The plant proteolytic machinery and its role in defence. Curr Opin Plant Biol 7:400–407
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034
Walker AJ, Urwin PE, Atkinson HJ, Brain P, Glen DM, Shewry PR (1999) Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Müller). Transgenic Res 8:95–103
Wang K-M, Kumar S, Cheng Y-S, Venkatagiri S, Yang AH, Yeh KW (2008) Characterization of inhibitory mechanism and antifungal activity between group-1 and group-2 phytocystatins from taro (Colocasia esculenta). FEBS J 275:4980–4989
Watanabe H, Abe K, Emori Y, Hosoyama H (1991) Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (oryzains). J Biol Chem 266:16897–16902
Zhang X, Liu S, Takano T (2008) Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance. Plant Mol Biol 68:131–143
Acknowledgments
This work was supported by CNPq Grant 478417/2012-8. R. Margis has a research fellowship 307868/2011-7 from CNPq and A. P. Christoff a CAPES Ph.D. fellowship.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by A. K. Tyagi.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
438_2014_892_MOESM2_ESM.tif
Supplementary material 2 (TIFF 5592 kb). Fig. S1. Phylogenetic tree of phytocystatins. The protein sequences of 26 plant and algae genomes were analyzed to reconstruct a phylogenetic tree with the Bayesian method. Only the common N-terminal gene sequences were used in the analysis. The branch supports represent the posterior probability values, and the three main groups are identified with circled numbers. Rice phytocystatins are highlighted in red
438_2014_892_MOESM3_ESM.tif
Supplementary material 3 (TIFF 4141 kb). Fig. S2. Rice phytocystatin gene expression patterns. a RNAseq libraries from root and leaf (Arenhart et al. 2013) were analyzed. The RPKM method was used to normalize the read numbers. b Microarray datasets from the Rice Oligonucleotide array were evaluated to identify the phytocystatin gene expression pattern through several plant development stages. Gene expression levels are measured according to the right-hand scale bar, on which yellow represents the more expressed genes. c RNAseq libraries from different rice tissues SRP008821 were mapped against the rice phytocystatins transcripts and the number of reads normalized with RPKM method. d RNAseq experiments from rice infected with compatible and incompatible blast fungus (Magnaporthe grisea), DRP000568. Reads were normalized using RPKM and letters above the error bars indicate statistical Kruskal–Wallis differences for each experimental triplicate
438_2014_892_MOESM4_ESM.tif
Supplementary material 4 (TIFF 739 kb). Fig. S3. Relative cysteine protease activity during rice germination. Papain and legumain protease activities were measured in total protein extracts (per mg of protein) from stages of dry seed (0), 0.5, 1, 2, 4, and 8 days after the beginning of plant germination. Rice samples from 0, 0.5, 1, 2, 4 and 8 dag were collected in triplicate, frozen and macerated in liquid nitrogen. The ground material was thawed on ice and mixed with 1 mL of extraction buffer per 100 mg of sample. Papain-like activities were measured at 410 nm, with 2 mM Bz-DL-Arg-βNA-HCl colorimetric substrate (BACHEM). The extraction buffer was a phosphate buffer pH 6.0, containing 1 mM DTT and 2 mM EDTA. Legumain proteinase activity was measured with 1 mM Z-Ala-Ala-Asn-AMC fluorigenic substrate (BACHEM) in an extraction buffer of citrate–phosphate pH 5.4, containing 1 mM DTT and 2 mM EDTA. Free AMC release was measured at excitation and emission wavelengths 360 and 460 nm, respectively. The protein concentration of the samples was determined in triplicate with a serial Bradford dilution (Bradford 1976). These data represent the results of three independent experiments. The replicate averages were compared using an ANOVA one-way statistical test and Duncan’s test; significance set at p < 0.05
Rights and permissions
About this article
Cite this article
Christoff, A.P., Margis, R. The diversity of rice phytocystatins. Mol Genet Genomics 289, 1321–1330 (2014). https://doi.org/10.1007/s00438-014-0892-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-014-0892-7