Abstract
Key message
Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity.
Abstract
Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.
Similar content being viewed by others
Abbreviations
- APs:
-
Aspartic proteases
- BSA:
-
Bovine serum albumin
- HR:
-
Hairy roots
- PBS:
-
Phosphate-buffered saline
- PMSF:
-
Phenylmethylsulfonyl fluoride
- PVPP:
-
Polyvinylpolypyrrolidone
- SH:
-
Schenk and Hildebrandt medium
References
Adamczyk B, Godlewski M, Smolander A, Kitunen V (2009) Degradation of proteins by enzymes exuded by Allium porrum roots-A potentially important strategy for acquiring organic nitrogen by plants. Plant Physiol Biochem 47:919–925. https://doi.org/10.1016/j.plaphy.2009.05.010
Agostini E, de Forchetti SM, Tigier HA (1997) Production of peroxidases by hairy roots of Brassica napus. Plant Cell Tissue Organ Cult 47:177–182. https://doi.org/10.1007/BF02318955
Almeida CM, Simões I (2018) Cardoon-based rennets for cheese production. Appl Microbiol Biotechnol 102:4675–4686. https://doi.org/10.1007/s00253-018-9032-3
Amselem J, Tepfer M (1992) Molecular basis for novel root phenotypes induced by Agrobacterium rhizogenes A4 on cucumber. Plant Mol Biol 19:421–432. https://doi.org/10.1007/BF00023390
Asakura T, Watanabe H, Abe K, Arai S (1997) Oryzasin as an aspartic proteinase occurring in rice seeds: purification, characterization, and application to milk clotting. J Agric Food Chem 45:1070–1075. https://doi.org/10.1021/jf960582x
Azuma N, Kaminogawa S, Yamauchi K (1984) Properties of glycomacropeptide and para-k-casein derived from human k-casein and comparison of human and bovine k-caseins as to susceptibility to chymosin and pepsin. Agric Biol Chem 48:2025–2031. https://doi.org/10.1080/00021369.1984.10866443
Bull CT, De Boer SH, Denny TP, Firrao G, Fischer-Le Saux M, Saddler GS, Scortichini M, Stead DE, Takikawa Y (2010) Comprehensive list of names of plant pathogenic bacteria, 1980–2007. J Plant Pathol 92:551–592. https://doi.org/10.4454/JPP.V96I2.026
Cercós M, Urbez C, Carbonell J (2003) A serine carboxypeptidase gene (PsCP), expressed in early steps of reproductive and vegetative development in Pisum sativum, is induced by gibberellins. Plant Mol Biol 51:165–174. https://doi.org/10.1023/A:1021142403856
D’Ambrosio A, Rossano R, Ungaro N, Riccio P (2003) Proteolytic and milk clotting activities in extracts obtained from the crustaceans Munida. J Mol Catal B Enzym 22:145–150. https://doi.org/10.1016/S1381-1177(03)00026-2
Davies LJ, Zhang L, Elling AA (2015) The Arabidopsis thaliana papain-like cysteine protease RD21 interacts with a root-knot nematode effector protein. Nematology 17:655–666. https://doi.org/10.1163/15685411-00002897
Drøhse HB, Foltmann B (1989) Specificity of milk-clotting enzymes towards bovine κ-casein. Biochim Biophys Acta-Protein Struct Mol Enzymol 995:221–224. https://doi.org/10.1016/0167-4838(89)90039-3
Duarte P, Pissarra J, Moore I (2008) Processing and trafficking of a single isoform of the aspartic proteinase cardosin a on the vacuolar pathway. Planta 227:1255–1268. https://doi.org/10.1007/s00425-008-0697-1
Faro C, Gal S (2005) Aspartic proteinase content of the arabidopsis genome. Curr Protein Pept Sci 6:493–500. https://doi.org/10.2174/13890305774933268
Faro C, Ramalho-Santos M, Vieira M, Mendes A, Simões I, Andrade R, Verissimo P, Lin X, Tang J, Pires E (1999) Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase. J Biol Chem 274:28724–28729. https://doi.org/10.1074/jbc.274.40.28724
Geerlings A, Hallard D, Caballero AM, Cardoso IL, Van der Heijden R, Verpoorte R (1999) Alkaloid production by a Cinchona officinalis “Ledgeriana” hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus. Plant Cell Rep 19:191–196. https://doi.org/10.1007/s002990050732
Hegelund JN, Lauridsen UB, Wallström SV, Muller R, Lutken H (2017) Transformation of Campanula by wild type Agrobacterium rhizogenes. Euphytica 213:51. https://doi.org/10.1007/s10681-017-1845-0
Heimgartner U, Pietrzak M, Geertsen R, Brodelius P, Figueiredo AC, Pais MSS (1990) Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus. Phytochemistry 29:1405–1410. https://doi.org/10.1016/0031-9422(90)80090-4
Juge N (2006) Plant protein inhibitors of cell wall degrading enzymes. Trens Plant Sci 7:359–367. https://doi.org/10.1016/j.tplants.2006.05.006
Kim YH, Yoo YJ (1996) Peroxidase production from carrot hairy root cell culture. Enzym Microb Technol 18:531–535. https://doi.org/10.1016/0141-0229(95)00168-9
Kohli A, Narciso JO, Miro B, Raorane M (2012) Root proteases: reinforced links between nitrogen uptake and mobilization and drought tolerance. Physiol Plant 145:165–179. https://doi.org/10.1111/j.1399-3054.2012.01573.x
Lane CN, Fox PF, Johnston DE, McSweeney PLH (1997) Contribution of coagulant to proteolysis and textural changes in cheddar cheese during ripening. Int Dairy J 7:453–464. https://doi.org/10.1016/S0958-6946(97)00041-1
Lehfeldt C, Shirley AM, Meyer K, Ruegger MO, Cusumano JC, Viitanen PV, Strack D, Chapple C (2000) Cloning of the SNG1 gene of arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12:1295–1306. https://doi.org/10.1105/tpc.12.8.1295
Li J, Lease KA, Tax FE, Walker JC (2001) BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana. PNAS 98:5916–5921. https://doi.org/10.1073/pnas.091065998
Lima Costa ME, Van Gulik WM, Ten Hoopen JG, Pais MSS, Cabral JMS (1996) Protease and phenol production of Cynara cardunculus L. Cell suspension in a chemostat. Enzyme Microb Technol 19:493–500. https://doi.org/10.1016/S0141-0229(96)00058-0
Liu H, Wang X, Zhang H, Yang Y, Ge X, Song F (2008) A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress. Gene 420:57–65. https://doi.org/10.1016/j.gene.2008.05.006
Lourenço PML, de Castro S, Martins TM, Clemente A, Domingos A (2002) Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose. Enzyme Microb Technol 31:242–249. https://doi.org/10.1016/S0141-0229(02)00117-5
Mano Y, Ohkawa H, Yamada Y (1989) Production of tropane alkaloids by hairy root cultures of Duboisia leichhardtii transformed by Agrobacterium rhizogenes. Plant Sci 59:191–201. https://doi.org/10.1016/0168-9452(89)90137-4
Martins APL, De Vasconcelos MMP, De Sousas RB (1996) Thistle (Cynara cardunculus L) flower as a coagulant agent for cheesemaking. Short characterization. Lait 76:473–477. https://doi.org/10.1051/lait:1996536
Moore L, Warren G, Strobel G (1979) Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid 2:617–626. https://doi.org/10.1016/0147-619X(79)90059-3
Oliveira A, Pereira C, da Costa DS et al (2010) Characterization of aspartic proteinases in C. cardunculus L. callus tissue for its prospective transformation. Plant Sci 178:140–146. https://doi.org/10.1016/j.plantsci.2009.11.008
Pereira CS, Da Costa DS, Pereira S, Moura Nogueira F, Albuquerque PM, Teixeira J, Faro C, Pissarra J (2008) Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases. Protoplasma 232:203–213. https://doi.org/10.1007/s00709-008-0288-9
Pimentel C, Van Der Straeten D, Pires E, Faro C, Rodrigues-Pousada C (2007) Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L. FEBS J 274:2523–2539. https://doi.org/10.1111/j.1742-4658.2007.05787.x
Pires AS, Cabral MG, Fevereiro P, Stoger E, Abranches R (2008) High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures. Biotechnol J 3:916–923. https://doi.org/10.1002/biot.200800044
Ramos PAB, Guerra ÂR, Guerreiro O, Santos SAO, Oliveira H, Freire CSR, Silvestre AJD, Duarte MF (2017) Antiproliferative effects of Cynara cardunculus L. Var. altilis (DC) lipophilic extracts. Int J Mol Sci 18:63. https://doi.org/10.3390/ijms18010063
Raposo S, Domingos A (2008) Purification and characterization milk-clotting aspartic proteinases from Centaurea calcitrapa cell suspension cultures. Process Biochem 43:139–144. https://doi.org/10.1016/j.procbio.2007.11.003
Roseiro LB, Barbosa M, Ames JM, Wilbey RA (2003) Cheesemaking with vegetable coagulants-the use of Cynara cardunculus L. for the production of ovine milk cheeses. Int J Dairy Technol 56:76–85. https://doi.org/10.1046/j.1471-0307.2003.00080.x
Santos RB, Abranches R, Fischer R, Sack M, Holland T (2016) Putting the spotlight back on plant suspension cultures. Front Plant Sci 7:1–12. https://doi.org/10.3389/fpls.2016.00297
Santos RB, Chandrasekar B, Mandal MK, Kashani F, Kaiser M, Both L, Van der Hoorn RAL, Schiermeyer A, Abranches R (2018) Low protease content in Medicago truncatula cell cultures facilitates recombinant protein production. Biotechnol J 13:1–11. https://doi.org/10.1002/biot.201800050
Sarmento AC, Lopes H, Oliveira CS, Vitorino R, Samyn B, Sergeant K, Debyser G, Van Beeumen J, Domingues P, Amado F, Pires E, Domingues MRM, Barros MT (2009) Multiplicity of aspartic proteinases from Cynara cardunculus L. Planta 230:429–439. https://doi.org/10.1007/s00425-009-0948-9
Schaller A (2004) A cut above the rest: the regulatory function of plant proteases. Planta 220:183–197. https://doi.org/10.1007/s00425-004-1407-2
Sharma P, Padh H, Shrivastava N (2013) Hairy root cultures: a suitable biological system for studying secondary metabolic pathways in plants. Eng Life Sci 13:62–75. https://doi.org/10.1002/elsc.201200030
Shindo T, Misas-Villamil JC, Hörger AC, Song J, van der Hoorn RAL (2012) A role in immunity for arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14. PLOS One 7(1):e29317. https://doi.org/10.1371/journal.pone.0029317
Simões I, Faro R, Bur D, Faro C (2004) Structure and function of plant aspartic proteinases. Eur J Biochem 271:2067–2075. https://doi.org/10.1111/j.1432-1033.2004.04136.x
Singh TK, Young ND, Drake MA, Cadwallader KR (2005) Production and sensory characterization of a bitter peptide from β-casein. J Agric Food Chem 53:1185–1189. https://doi.org/10.1021/jf049058d
Taya M, Yoyama A, Nomura R, Kondo O, Matsui C, Kobayashi T (1989) Production of peroxidase with horseradish hairy root cells in a two step culture system. J Ferment Bioeng 67:31–34. https://doi.org/10.1016/0922-338X(89)90082-2
Verissimo P, Faro C, Moir AJG, Lin Y, Tang J, Pires E (1996) Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L. Eur J Biochem 235:762–768. https://doi.org/10.1111/j.1432-1033.1996.00762.x
Veríssimo P, Esteves C, Faro C, Pires E (1995) The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities. Biotechnol Lett 17:621–626. https://doi.org/10.1007/BF00129389
Vieira M, Pissarra J, Veríssimo P, Castanheira P, Costa Y, Pires E, Faro C (2001) Molecular cloning and characterization of cDNA encoding cardosin B, an aspartic proteinase accumulating extracellularly in the transmitting tissue of Cynara cardunculus L. Plant Mol Biol 45:529–539. https://doi.org/10.1023/A:1010675015318
Zhu LH, Holefors A, Ahlman A, Xue TZ, Welander M (2001) Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth. Plant Sci 160:433–439. https://doi.org/10.1016/S0168-9452(00)00401-5
Acknowledgements
This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through project “Proteases from Cynara cardunculus L.: study of gene expression and establishment of alternative production platforms” (ref. PTDC/BAA-AGR/30447/2017), Research Unit Bioresources 4 Sustainability (ref. UID/Multi/04551/2013), and PhD Fellowship to AF (ref. PD/BD/114488/2016, Plants for Life PhD Program). MS data were obtained by the UniMS – Mass Spectrometry Unit, ITQB/IBET, Oeiras, Portugal. The authors wish to thank Isaura Simões and Rita B. Santos for helpful discussions throughout the course of this work, Bruno Alexandre for help with the MS analysis, and Andreas Schiermeyer for critical reading of the manuscript.
Author information
Authors and Affiliations
Contributions
AF and RA conceived and designed the research. AF and ASP conducted experiments. ACF and CP contributed reagents and tools for hairy root cultures and gene expression analysis, respectively. AF and RA wrote the manuscript. All authors analyzed the data and approved the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Maike Petersen.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Folgado, A., Pires, A.S., Figueiredo, A.C. et al. Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.. Plant Cell Rep 39, 89–100 (2020). https://doi.org/10.1007/s00299-019-02475-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-019-02475-1