Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus

Industrial chicory (Cichorium intybus var. sativum) and witloof (C. intybus var. foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway are GERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO), COSTUNOLIDE SYNTHASE (COS) and KAUNIOLIDE SYNTHASE (KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found that C. intybus SL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybus CYP71 enzymes transiently produced in Nicotiana benthamiana and identified several functional paralogs for each of the GAO, COS and KLS genes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing in C. intybus. Metabolite profiling of mutant C. intybus lines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into the C. intybus SL biosynthetic pathway and paves the way for the engineering of C. intybus bitterness.

[1]  N. Dauchot,et al.  Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility , 2023, bioRxiv.

[2]  D. Bosch,et al.  CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots , 2022, Frontiers in Plant Science.

[3]  B. Ren,et al.  Sesquiterpene lactones with anti-inflammatory and cytotoxic activities from the roots of Cichorium intybus. , 2022, Phytochemistry.

[4]  C. J. Inulin , 2022, Reactions Weekly.

[5]  Weiming He,et al.  The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis. , 2022, Genomics.

[6]  P. Dawyndt,et al.  Stack Mapping Anchor Points (SMAP): a versatile suite of tools for read-backed haplotyping , 2022, bioRxiv.

[7]  A. Goossens,et al.  Jasmonate: A hormone of primary importance for plant metabolism. , 2022, Current opinion in plant biology.

[8]  K. Oksman-Caldentey,et al.  Chicory Extracts and Sesquiterpene Lactones Show Potent Activity against Bacterial and Fungal Pathogens , 2021, Pharmaceuticals.

[9]  D. Bosch,et al.  Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L. , 2021, Plant biotechnology journal.

[10]  C. Santos,et al.  Sesquiterpene Lactones: Promising Natural Compounds to Fight Inflammation , 2021, Pharmaceutics.

[11]  B. Olas,et al.  The Plants of the Asteraceae Family as Agents in the Protection of Human Health , 2021, International journal of molecular sciences.

[12]  T. Misaka,et al.  Characterization of the Human Bitter Taste Receptor Response to Sesquiterpene Lactones from Edible Asteraceae Species and Suppression of Bitterness through pH Control , 2021, ACS omega.

[13]  T. Ruttink,et al.  Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) , 2020, Frontiers in Genome Editing.

[14]  A. Goossens,et al.  Combinatorial Control of Plant Specialized Metabolism: Mechanisms, Functions, and Consequences. , 2020, Annual review of cell and developmental biology.

[15]  H. Bouwmeester,et al.  Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L. , 2019, GM crops & food.

[16]  P. Zerbe,et al.  Terpene Synthases as Metabolic Gatekeepers in the Evolution of Plant Terpenoid Chemical Diversity , 2019, Front. Plant Sci..

[17]  E. Schijlen,et al.  Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L. (Asteraceae) , 2019, Industrial Crops and Products.

[18]  V. Guallar,et al.  Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity , 2018, Nature Communications.

[19]  M. Maffei,et al.  TPS Genes Silencing Alters Constitutive Indirect and Direct Defense in Tomato , 2018, International journal of molecular sciences.

[20]  J. Hodson,et al.  Silencing amorpha-4,11-diene synthase Genes in Artemisia annua Leads to FPP Accumulation , 2018, Front. Plant Sci..

[21]  Yves Van de Peer,et al.  PLAZA 4.0: an integrative resource for functional, evolutionary and comparative plant genomics , 2017, Nucleic Acids Res..

[22]  C. Wasternack,et al.  Jasmonates are signals in the biosynthesis of secondary metabolites - Pathways, transcription factors and applied aspects - A brief review. , 2017, New biotechnology.

[23]  P. Clercq,et al.  Induced expression of selected plant defence related genes in pot azalea, Rhododendron simsii hybrid , 2017, Euphytica.

[24]  Anne Osbourn,et al.  A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules , 2017, Metabolic engineering.

[25]  Xun Xu,et al.  Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce , 2017, Nature Communications.

[26]  M. Gonnella,et al.  Insights into the Sesquiterpenoid Pathway by Metabolic Profiling and De novo Transcriptome Assembly of Stem-Chicory (Cichorium intybus Cultigroup “Catalogna”) , 2016, Front. Plant Sci..

[27]  G. Barcaccia,et al.  Current Advances in Genomics and Breeding of Leaf Chicory ( Cichorium intybus L.) , 2016 .

[28]  F. Ferioli,et al.  The impact of sesquiterpene lactones and phenolics on sensory attributes: An investigation of a curly endive and escarole germplasm collection. , 2016, Food chemistry.

[29]  S. Jackson,et al.  Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce , 2016, Plant Methods.

[30]  M. Abdollahi,et al.  Sesquiterpene lactone engineering in microbial and plant platforms: parthenolide and artemisinin as case studies , 2016, Applied Microbiology and Biotechnology.

[31]  O. Maudoux,et al.  Exploration of genetic diversity within Cichoriumendivia and Cichorium intybus with focus on the gene pool of industrial chicory , 2016, Genetic Resources and Crop Evolution.

[32]  F. D. da Costa,et al.  Sesquiterpene Lactones: More Than Protective Plant Compounds With High Toxicity , 2016 .

[33]  B. Ivanescu,et al.  Sesquiterpene Lactones from Artemisia Genus: Biological Activities and Methods of Analysis , 2015, Journal of analytical methods in chemistry.

[34]  Wangzhen Guo,et al.  The cytochrome P450 superfamily: Key players in plant development and defense , 2015 .

[35]  D. Gagneul,et al.  Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus , 2015, Front. Plant Sci..

[36]  L. F. D'Antuono,et al.  Variation of sesquiterpene lactones and phenolics in chicory and endive germplasm , 2015 .

[37]  A. Beharav,et al.  Variation of sesquiterpene lactone contents in Lactuca georgica natural populations from Armenia , 2015, Genetic Resources and Crop Evolution.

[38]  A. Osbourn,et al.  OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua , 2015, The Plant Cell.

[39]  R. D. de Vos,et al.  Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway. , 2014, Metabolic engineering.

[40]  V. Galli,et al.  Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses , 2014, Planta.

[41]  C. Wagstaff,et al.  Sesquiterpenoids Lactones: Benefits to Plants and People , 2013, International journal of molecular sciences.

[42]  Yves Van de Peer,et al.  ORCAE: online resource for community annotation of eukaryotes , 2012, Nature Methods.

[43]  Ling-Jian Wang,et al.  The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L. , 2012, Molecular plant.

[44]  M. Sanderson,et al.  Seasonal variation in sesquiterpene lactone concentration and composition of forage chicory (Cichorium intybus L.) cultivars , 2011 .

[45]  M. Goedbloed,et al.  Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana , 2011, PloS one.

[46]  Marcel Martin Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[47]  D. Ro,et al.  Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism* , 2011, The Journal of Biological Chemistry.

[48]  W. Cui,et al.  Biotechnological potential of inulin for bioprocesses. , 2011, Bioresource technology.

[49]  Robert A. Edwards,et al.  Quality control and preprocessing of metagenomic datasets , 2011, Bioinform..

[50]  D. Bosch,et al.  A chicory cytochrome P450 mono‐oxygenase CYP71AV8 for the oxidation of (+)‐valencene , 2011, FEBS letters.

[51]  D. Ro,et al.  Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae* , 2010, The Journal of Biological Chemistry.

[52]  M. Robinson,et al.  A scaling normalization method for differential expression analysis of RNA-seq data , 2010, Genome Biology.

[53]  Davis J. McCarthy,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[54]  G. Lomonossoff,et al.  pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. , 2009, Plant biotechnology journal.

[55]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[56]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[57]  Bart Nicolai,et al.  Predicting sensory attributes of different chicory hybrids using physico-chemical measurements and visible/near infrared spectroscopy , 2008 .

[58]  G. Mortier,et al.  qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data , 2007, Genome Biology.

[59]  P. Nguyen-Dinh,et al.  Antimalarial activity of lactucin and lactucopicrin: sesquiterpene lactones isolated from Cichorium intybus L. , 2004, Journal of ethnopharmacology.

[60]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[61]  H. Bouwmeester,et al.  Isolation and Characterization of Two Germacrene A Synthase cDNA Clones from Chicory1 , 2002, Plant Physiology.

[62]  M. Joerink,et al.  Biosynthesis of Costunolide, Dihydrocostunolide, and Leucodin. Demonstration of Cytochrome P450-Catalyzed Formation of the Lactone Ring Present in Sesquiterpene Lactones of Chicory , 2002, Plant Physiology.

[63]  H. Bouwmeester,et al.  Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis. , 2001, Plant physiology.

[64]  A. Drewnowski,et al.  Bitter taste, phytonutrients, and the consumer: a review. , 2000, The American journal of clinical nutrition.

[65]  M. Bennett,et al.  Metabolite profiling of sesquiterpene lactones from Lactuca species. Major latex components are novel oxalate and sulfate conjugates of lactucin and its derivatives. , 2000, The Journal of biological chemistry.

[66]  T. Mes,et al.  A search for diagnostic AFLP markers in Cichorium species with emphasis on endive and chicory cultivar groups , 2000 .

[67]  J. E. Oltra,et al.  New sources and antifungal activity of sesquiterpene lactones. , 2000, Fitoterapia.

[68]  H. Bouwmeester,et al.  (+)-Germacrene A biosynthesis . The committed step in the biosynthesis of bitter sesquiterpene lactones in chicory , 1998, Plant physiology.

[69]  T. V. van Beek,et al.  Bitter sesquiterpene lactones from chicory roots , 1990 .

[70]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[71]  Laurence Desmet,et al.  A General Protocol for Accurate Gene Expression Analysis in Plants. , 2020, Methods in molecular biology.

[72]  Lies Kips,et al.  Characterization and processing of horticultural byproducts : a case study of tomato and Belgian endive roots , 2017 .

[73]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[74]  T. Mes,et al.  A search for diagnostic AFLP markers in Cichorium species with emphasis on endive and chicory cultivar groups. , 2000, Genome.