A putative serine protease with a potential role in the plant biotic and abiotic stress response was purified from wheat leaf apoplastic fluid and partially characterized. Following two-dimensional electrophoresis a protein of Mr = 75 k and a pI of 4.2 to 4.5 was observed. This protein displayed in-gel protease activity and was specifically inhibited by phenylmethanesulfonyl fluoride and partially inhibited by Ca2+ and Zn2+, but not by E-64 or leupeptin. An internal tryptic fragment of 13 amino acids was identified by MALDI QqTOF MS/MS, and this peptide showed a high level of homology (85–100 % identity) to a highly conserved region of known plant subtilisin-like proteases. We demonstrated that the protease activity increased until a late stage of wheat leaf development and increased in response to heat shock. In both cases Rubisco large subunit was degraded with time. Protease activity was also increased during biotic stress. Leaves challenged with leaf rust (Puccinia triticina), showed an approximately three fold increase in protease activity during an incompatible interaction, compared to activity in mock-inoculated leaves and to leaves in a compatible leaf rust interaction. These results suggest that the expression of this serine protease could be involved in the defense response against both abiotic and biotic stresses.
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effector triggered immunity
matrix-assisted laser desorption/ionization
microbe-associated molecular patterns
tandem mass spectrometry
programmed cell death
pattern recognition receptors
Rubisco large subunit
reactive oxygen species
Beers, E. P., & McDowell, J. M. (2001). Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues. Current Opinion in Plant Biology, 4, 561–567.
Berger, D., & Altmann, T. (2000). A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes & Development, 14, 1119–1131.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.
Budic, M., Sabotic, J., Megic, V., Kos, J., & Kidric, M. (2013). Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought. Plant Physiology and Biochemistry, 62, 79–87.
Coffeen, W. C., & Wolpert, T. J. (2004). Purification and characterization of serine protease that exhibit caspase-like activity and are associated with programmed cell death in Avene sativa. The Plant Cell, 16, 857–873.
Coll, N. S., Epple, P., & Dangl, J. L. (2011). Programmed cell death in the plant immune system. Cell Death and Differentiation, 18, 1289–1297.
Dangl, J. L., & Jones, J. D. G. (2001). Plant pathogens and integrated defence responses to infection. Nature, 411, 826–833.
Dannel, F., Pfeffer, H., & Marschner, H. (1995). Isolation of apoplastic fluid from sunflower leaves and its use for studies on influence of nitrogen supply on apoplastic pH. Journal of Plant Physiology, 146, 273–278.
Desmond, O. J., Manners, J. M., Stephens, A. E., Maclean, D. J., Schenk, P. M., Gardiner, D. M., et al. (2008). The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat. Molecular Plant Pathology, 9, 435–445.
Dominguez, F., & Cejudo, F. J. (1996). Characterization of endoproteases appears during wheat grain development. Plant Physiology, 112, 1211–1217.
Fan, T., & Xing, T. (2004). Heat shock induces programmed cell death in wheat leaves. Biologia Plantarum, 48, 389–394.
Fan, T., Gao, Y., Al-Shammari, A., Wang, X. J., & Xing, T. (2009). Yeast two-hybrid screening of MAP kinase cascade identifies cytosolic glutamine synthetase 1b as a tMEK2 interactive protein in wheat. Canadian Journal of Plant Pathology, 31, 407–414.
Feller, U., Anders, I., & Mae, T. (2008). Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated. Journal of Experimental Botany, 59, 1615–1624.
Flor, H. H. (1971). Current status of the gene-for-gene concept. Annual Review of Phytopathology, 9, 275–296.
Gao, Y., Liu, X. W., Stebbing, J., He, D. F., Laroche, A., Gaudet, D., & Xing, T. (2011). TaFLRS, a novel mitogen-activated protein kinase in wheat defense responses. European Journal of Plant Pathology, 131, 643–651.
Glazebrook, J. (2001). Genes controlling expression of defense responses in Arabidopsis – 2001 status. Current Opinion in Plant Biology, 4, 301–308.
Greenberg, J. T. (1996). Programmed cell death: a way of life for plants. Proceedings of the National Academy of Sciences of the United States of America, 93, 12094–12097.
Heussen, C., & Dowdle, E. (1980). Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates. Analytical Biochemistry, 102, 198–202.
Holwerda, B. C., & Rogers, J. C. (1992). Purification and characterization of aleurain. Plant Physiology, 99, 848–855.
Hu, G., & Rijkenberg, F. H. J. (1998). Scanning electron microscopy of early infection structure formation by Puccinia recondite f. sp. Tritici on and in susceptible and resistant wheat lines. Mycology Research, 102, 391–399.
Jordá, L., Coego, A., Conejero, V., & Vera, P. (1999). A genomic cluster containing four differentially regulated subtilisin-like processing protease genes is in tomato plants. Journal of Biological Chemistry, 274, 2360–2365.
Jordan, M., Cloutier, S., Somers, D., Procunier, D., Rampitsch, C., & Xing, T. (2006). Beyond R genes: dissecting disease-resistance pathways using genomics and proteomics. Canadian Journal of Plant Pathology, 28S, 228–232.
Kolmer, J. A. (1996). Genetics of resistance to wheat leaf rust. Annual Review of Phytopathology, 34, 435–455.
Loboda, A. V., Krutchinsky, A. N., Bromirski, M., Ens, W., & Standing, K. G. (2000). A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance. Rapid Communications in Mass Spectrometry, 14, 1047–1057.
Plett, J. M., Cvetkovska, M., Makenson, P., Xing, T., & Regan, S. (2009). Arabidopsis ethylene receptors have different roles in Fumonisin B1-induced cell death. Physiological and Molecular Plant Pathology, 74, 18–26.
Ramirez, V., Lopez, A., Mauch-Mani, B., Gil, M., & Vera, P. (2013). An extra cellular subtilase switch for immune priming in Arabidopsis. PLoS Pathogen, 9, e1003445. doi:10.1371/journal.ppat.1003445.
Rampitsch, C., Bykova, N. V., McCallum, B., Beimcik, E., & Ens, W. (2006). Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction. Proteomics, 6, 1897–1907.
Rantong, G., & Gunawardena, A. H. L. A. N. (2015). Programmed cell death: genes involved in signaling, regulation, and execution in plants and animals. Botany, 93, 193–210.
Rautergarten, C., Steinhauser, D., Bussis, D., Stintzi, A., Schaller, A., Kopka, J., et al. (2005). Inferring hypothesis on functional relationships of genes: analysis of the Arabidopsis thaliana subtilase gene family. PLoS Computational Biology, 1, 297–312.
Roberts, I. N., Murray, P. F., Caputo, C. P., Passeron, S., & Barneix, A. J. (2003). Purification and characterization of a subtilisin-like serine proteases induced during the senescence of wheat leaves. Physiologia Plantarum, 118, 453–490.
Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., et al. (1996). Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proceedings of the National Academy of Sciences of the United States of America, 93, 14440–14445.
Siezen, R. J., & Leunissen, J. A. M. (1997). Subtilases: The superfamily of subtilisin-like serine proteases. Protein Science, 6, 501–523.
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, 431–443.
Tao, Y., Xie, Z., Chen, W., Glazebrook, J., Chang, H.S., Han, B., et al. (2003). Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae. Plant Cell 15, 317–330.
Tornero, P., Coneejero, V., & Vera, P. (1997). Identification of a new pathogen-induced member of the subtilisin-like processing protease family from plants. Journal of Biological Chemistry, 272, 14412–14419.
Tripathi, L., & Sowdhamini, R. (2006). Cross genome comparisons of serine proteases in Arabidopsis and rice. BMC Genomics, 7, 200. doi:10.1186/1471-2164-7-200.
van der Hoorn, R. A. L. (2008). Plant proteases: from phenotypes to molecular mechanisms. Annual Review of Plant Biology, 59, 191–223.
van Loon, L. C., & van Strien, E. A. (1999). The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 55, 85–97.
van Wees, S. C., Chang, H. S., Zhu, T., & Glazebrook, J. (2003). Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling. Plant Physiology, 132, 606–617.
Vartapetian, A. B., Tuzhikov, A. I., Chichkova, N. V., Taliansky, M., & Wolpert, T. J. (2011). A plant alternative to animal caspases: subtilisin-like proteases. Cell Death and Differentiation, 18, 1289–1297.
Wan, L., Xia, Q., Qiu, X., & Selvaraj, G. (2002). Early stages of seed development in Brassica napus: a seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument. Plant Journal, 30, 1–10.
Xing, T. (2007). Signal transduction pathways and disease resistant genes and their applications to fungal disease control. In Z. K. Punja, S. H. Be Boer, & H. Sanfaçon (Eds.), Biotechnology and Plant Disease Management (pp. 1–15). UK: CAB International.
Xing, T., Fan, T., Han, S., Djuric-Ciganovic, S., Jordan, M. C., & Wang, X. J. (2005). Programmed cell death in plant disease resistance: Pathways and components. Recent Research Developments in Bioenergetics, 3, 33–44.
Thanks to Ken Standing and Werner Ens in University of Manitoba for generous access to their MALDI-QqTOF prototype mass spectrometer and to Brent McCallum, AAFC for providing the rust races. This work was funded by an internal grant from AAFC to TX and CR.
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Fan, T., Bykova, N.V., Rampitsch, C. et al. Identification and characterization of a serine protease from wheat leaves. Eur J Plant Pathol 146, 293–304 (2016). https://doi.org/10.1007/s10658-016-0914-x
- Subtilisin-like protease
- Programmed cell death