Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus
Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.
KeywordsCynara cardunculus Germacrene A synthase Single nucleotide polymorphisms Cynaropicrin Biological activities Association analysis
Cynara cardunculus L.
Electron impact mass spectra
Gas chromatography–mass spectrometry
Germacrene A synthase
High-Performance Liquid Chromatography–Ultraviolet-Visible analysis
Methicillin-Resistant Staphylococcus aureus
Minimum bactericidal concentration
Minimal inhibitory concentration
Minor allele frequency
- PROC GLM
Procedure general linear model
Plant seeds were kindly provided by Botanical Garden of University of Tuscia (Italy), Botanical Garden of University of Agronomic Sciences of Gembloux (Belgium), Botanical Garden of University of Szeged (Hungary), Botanical Garden of University of Napoli (Italy), Experimental Centre of School of Agriculture of Beja (Portugal) and Botanical Garden of Sapienza University of Rome (Italy). We are also grateful to the Horticultural Centre of School of Agriculture (ESA) of Beja, for space to grow Cc plants. Pseudomonas aeruginosa POAI was kindly donated by Professor Arsénio Fialho from Instituto Superior Técnico; Methicillin-resistant Staphylococcus aureus (clinical isolate from wound exudate) and Bacillus cereus were both kindly donated by Professor Manuela Pintado from Universidade Católica Portuguesa. We also thank Conceição Fernandes for HPLC support, Inês Pires for TASSEL software support and Anabel Usié Chimenos and Marcos Ramos for bioinformatic support. Fundação para a Ciência e a Tecnologia (FCT) is gratefully acknowledged for the PhD fellowships, refs. SFRH/BD/68847/2010; SFRH/BD/70845/2010, SFRH/BD/84406/2012, SFRH/BD/98635/2013 and SFRH/BD/110969/2015 awarded to AF, PR, OG, AG and TB, respectively. SG was supported by Programa Ciência 2008, also funded by POPH (QREN). This project was funded by European Regional Development Fund (FEDER), ValBioTecCynara (ALT20-03-0145-FEDER-000038)—Economic valorization of Cardoon (Cynara cardunculus): study of natural variability and biotechnological applications. Thanks are also due to GREEN-it “Bioresources for sustainability” (UID/Multi/04551/2013). The authors also thank Junta de Andalucía (grant P12-AGR-1482) and Campus de Excelencia Internacional Agroalimentario (CeiA3) for supporting this research collaboration.
AMF performed the experimental work and carried out the bioinformatic data analysis. PR contributed for GC-MS and GC-FID planning. AG contributed for antithrombin and antiproliferative activities and PP contributed for the antimicrobial activity. TB helped in the preparation of leaf lipophilic-derived extracts and cynaropicrin quantification by HPLC. OG and EJ contributed for statistical analysis. CC, JC and FJY-L contributed for HRM planning and execution. MFD, MMO, SG and RL contributed for the research planning, funding, discussions and paper writing. All authors read and approved the final manuscript
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Human and animal studies
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
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