Transcriptome driven characterization of curly- and smooth-leafed endives reveals molecular differences in the sesquiterpenoid pathway

Abstract

Endives (Cichorium endivia L.) are popular vegetables, diversified into curly/frisée- and smooth/broad-leafed (escaroles) cultivar types (cultigroups), and consumed as fresh and bagged salads. They are rich in sesquiterpene lactones (STL) that exert proven function on bitter taste and human health. The assembly of a reference transcriptome of 77,022 unigenes and RNA-sequencing experiments were carried out to characterize the differences between endives and escaroles at the gene structural and expression levels. A set of 3177 SNPs distinguished smooth from curly cultivars, and an SNP-supported phylogenetic tree separated the cultigroups into two distinct clades, consistently with the botanical varieties of origin (crispum and latifolium, respectively). A pool of 699 genes maintained differential expression pattern (core-DEGs) in pairwise comparisons between curly vs smooth cultivars grown in the same environment. Accurate annotation allowed the identification of 26 genes in the sesquiterpenoid biosynthesis pathway, which included several germacrene A synthase, germacrene A oxidase and costunolide synthase members (GAS/GAO/COS module), required for the synthesis of costunolide, a key precursor of lactucopicrin- and lactucin-like sesquiterpene lactones. The core-DEGs contained a GAS gene (contig83192) that was positively correlated with STL levels and recurrently more expressed in curly than smooth endives, suggesting a cultigroup-specific behavior. The significant positive correlation of GAS/GAO/COS transcription and STL abundance (2.4-fold higher in frisée endives) suggested that sesquiterpenoid pathway control occurs at the transcriptional level. Based on correlation analyses, five transcription factors (MYB, MYB-related and WRKY) were inferred to act on contig83192/GAS and specific STL, suggesting the occurrence of two distinct routes in STL biosynthesis.

References

[1] Lucchin, M., Varotto, S., Barcaccia, G. & Parrini, P. in Handbook of Plant Breeding (eds Prohens, J. & Nuez, F.) 3-48 (2008).

[2] Rick, C. M. Chicory-endive hybridized: isolation necessary to prevent production of undesired hybrids by the two species. Calif. Agric. (Berkeley) 7, 7- 7 (1953).

[3] Bernardes, E. C., Benko-Iseppon, A. M., Vasconcelos, S., Carvalho, R. & Brasileiro-Vidal, A. C. Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae). Genet. Mol. Biol. 36, 357-363 (2013).

[4] De Simone, M., Morgante, M., Lucchin, M., Parrini, P. & Marocco, A. A first linkage map of Cichorium intybus L. using a one-way pseudo-testcross and PCR-derived markers. Mol. Breed. 3, 415-425 (1997).

[5] Galla, G., Ghedina, A., Tiozzo, S. C. & Barcaccia, G. in Plant Genomics (ed. Abdurakhmonov, I. Y.) 67-87 (InTech, Janeza Trdine, Croatia 2016).

[6] Bai, C., Alverson, W. S., Follansbee, A. & Waller, D. M. New reports of nuclear DNA content for 407 vascular plant taxa from the United States. Ann. Bot. 110, 1623-1629 (2012).

[7] Cadalen, T. et al. Development of SSR markers and construction of a consensus genetic map for chicory (Cichorium intybus L.). Mol. Breed. 25, 699-722 (2010).

[8] Hodgins, K. A. et al. Genomics of Compositae crops: reference transcriptome assemblies and evidence of hybridization with wild relatives. Mol. Ecol. Resour. 14, 166-177 (2014).

[9] Ryder, E. J. Lettuce, Endive and Chicory Vol. 9 (CABI Publishing, New York, NY 1999).

[10] Chadwick, M., Trewin, H., Gawthrop, F. & Wagstaff, C. Sesquiterpenoids lactones: benefits to plants and people. Int. J. Mol. Sci. 14, 12780-12805 (2013).

[11] Amorim, M. H. R., da Costa, R. M. G., Lopes, C. & Bastos, M. M. S. M. Sesquiterpene lactones: adverse health effects and toxicity mechanisms. Crit. Rev. Toxicol. 43, 559-579 (2013).

[12] Ferioli, F., Manco, M. A. & D’Antuono, L. F. Variation of sesquiterpene lactones and phenolics in chicory and endive germplasm. J. Food Compost. Anal. 39, 77-86 (2015).

[13] Mai, F. & Glomb, M. A. Structural and sensory characterization of novel sesquiterpene lactones from iceberg lettuce. J. Agric. Food Chem. 64, 295-301 (2016).

[14] Bischoff, T. A. et al. Antimalarial activity of lactucin and lactucopicrin: sesquiterpene lactones isolated from Cichorium intybus L.. J. Ethnopharmacol. 95, 455-457 (2004).

[15] Wesolowska, A., Nikiforuk, A., Michalska, K., Kisiel, W. & Chojnacka-Wojcik, E. Analgesic and sedative activities of lactucin and some lactucin-like guaianolides in mice. J. Ethnopharmacol. 107, 254-258 (2006).

[16] Venkatesan, R., Shim, W. S., Yeo, E. J. & Kim, S. Y. Lactucopicrin potentiates neuritogenesis and neurotrophic effects by regulating Ca²⁺/CaMKII/ATF1 signaling pathway . J. Ethnopharmacol. 198, 174-183 (2017).

[17] Drewnowski, A. & Gomez-Carneros, C. Bitter taste, phytonutrients, and the consumer: a review. Am. J. Clin. Nutr. 72, 1424-1435 (2000).

[18] Van Beek, T. A. et al. Bitter sesquiterpenes lactones from chicory roots. J. Agric. Food Chem. 38, 1035-1038 (1990).

[19] Chadwick, M., Gawthrop, F., Michelmore, R. W., Wagstaff, C. & Methven, L. Perception of bitterness, sweetness and liking of different genotypes of lettuce. Food Chem. 197, 66-74 (2016).

[20] Filippo D’Antuono, L., Ferioli, F. & Manco, M. A. The impact of sesquiterpene lactones and phenolics on sensory attributes: an investigation of a curly endive and escarole germplasm collection. Food Chem. 199, 238-245 (2016).

[21] de Kraker, J. W., Franssen, M. C., Joerink, M., de Groot, A. & Bouwmeester, H. J. Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome p450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory. Plant Physiol. 129, 257-268 (2002).

[22] de Kraker, J. W., Franssen, M. C., Dalm, M. C., de Groot, A. & Bouwmeester, H. J. 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. Plant Physiol. 125, 1930-1940 (2001).

[23] Bouwmeester, H. J. et al. Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiol. 129, 134-144 (2002).

[24] Testone, G. et al. Insights into the sesquiterpenoid pathway by metabolic profiling and de novo transcriptome assembly of stem-chicory (Cichorium intybus cultigroup “Catalogna”). Front. Plant Sci. 7, https://doi.org/10.3389/fpls.2016.01676 (2016).

[25] D’Acunzo, F. et al. Influence of cultivation sites on sterol, nitrate, total phenolic contents and antioxidant activity in endive and stem chicory edible products. Int. J. Food Sci. Nutr. 68, 52-64 (2016).

[26] Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644-652 (2011).

[27] Zerbino, D. R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821-829 (2008).

[28] Schulz, M. H., Zerbino, D. R., Vingron, M. & Birney, E. Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 28, 1086-1092 (2012).

[29] Pertea, G. et al. TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 19, 651-652 (2003).

[30] Li, W. & Godzik, A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 1658-1659 (2006).

[31] Koonin, E. V. et al. A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes. Genome Biol. 5, R7 (2004).

[32] Conesa, A. et al. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674-3676 (2005).

[33] Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27-30 (2000).

[34] Ye, J. et al. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293-W297 (2006).

[35] Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A. C. & Kanehisa, M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 35, W182-W185 (2007).

[36] Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236-1240 (2014).

[37] Jin, J. et al. PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res. 45, D1040-D1045 (2017).

[38] Simao, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210-3212 (2015).

[39] Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009).

[40] Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).

[41] Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729 (2013).

[42] Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012).

[43] Muller, P. Y., Janovjak, H., Miserez, A. R. & Dobbie, Z. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32, 1372-1374 (2002). 1376, 1378- 1379.

[44] Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010).

[45] R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria, 2017).

[46] Nguyen, T. D. et al. Discovery of germacrene A synthases in Barnadesia spinosa: the first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae. Biochem. Biophys. Res. Commun. 479, 622-627 (2016).

[47] Bennett, M. H., Mansfield, J. W., Lewis, M. J. & Beale, M. H. Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.). Phytochemistry 60, 255-261 (2002).

[48] Liu, Q. et al. Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana. PLoS ONE 6, e23255 (2011).

[49] Nguyen, T. D. et al. Biochemical conservation and evolution of germacrene A oxidase in asteraceae. J. Biol. Chem. 285, 16588-16598 (2010).

[50] Ikezawa, N. et al. Lettuce costunolide synthase (CYP71BL2) and its homolog (CYP71BL1) from sunflower catalyze distinct regio- and stereoselective hydroxylations in sesquiterpene lactone metabolism. J. Biol. Chem. 286, 21601-21611 (2011).

[51] Reyes-Chin-Wo, S. et al. Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce. Nat. Commun. 8, 14953 New York, NY (2017).

[52] Scaglione, D. et al. The genome sequence of the outbreeding globe artichoke constructed de novo incorporating a phase-aware low-pass sequencing strategy of F1 progeny. Sci. Rep. 6, 19427 (2016).

[53] Badouin, H. et al. The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution. Nature 546, 148-152 (2017).

[54] Kiers, A. M. in Gorteria. Supplement. Vol. 5 (ed. Universiteit Leiden Branch Nationaal Herbarium Nederland) 1-77 (Universiteit Leiden: Leiden, The Netherlands, 2000).

[55] Kiers, A. M., Mes, T. H., van der Meijden, R. & Bachmann, K. A search for diagnostic AFLP markers in Cichorium species with emphasis on endive and chicory cultivar groups. Genome 43, 470-476 (2000).

[56] Hodgins, K. A. et al. Comparative genomics in the Asteraceae reveals little evidence for parallel evolutionary change in invasive taxa. Mol. Ecol. 24, 2226-2240 (2015).

[57] Raulier, P., Maudoux, O., Notte, C., Draye, X. & Bertin, P. Exploration of genetic diversity within Cichorium endivia and Cichorium intybus with focus on the gene pool of industrial chicory. Genet. Resour. Crop Evol. 63, 243-259 (2016).

[58] Barcaccia, G., Ghedina, A. & Lucchin, M. Current Advances in Genomics and Breeding of Leaf Chicory (Cichorium intybus L.). Agriculture-Basel 6, https://doi.org/10.3390/agriculture6040050 (2016).

[59] Blumel, M., Dally, N. & Jung, C. Flowering time regulation in crops-what did we learn from Arabidopsis?. Curr. Opin. Biotechnol. 32, 121-129 (2015).

[60] Huang, M. et al. The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-beta-caryophyllene, is a defense against a bacterial pathogen. New Phytol. 193, 997-1008 (2012).

[61] Street, R. A., Sidana, J. & Prinsloo, G. Cichorium intybus: traditional uses, phytochemistry, pharmacology, and toxicology. Evid. Based Complement. Altern. Med. 2013, 579319 (2013).

[62] Eljounaidi, K. et al. Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis. Plant Sci. 239, 128-136 (2015).

[63] Vranova, E., Coman, D. & Gruissem, W. Structure and dynamics of the isoprenoid pathway network. Mol. Plant 5, 318-333 (2012).

[64] Schluttenhofer, C. & Yuan, L. Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol. 167, 295-306 (2015).

[65] Kroes, A., vanLoon, J. J. & Dicke, M. Density-dependent interference of aphids with caterpillar-induced defenses in Arabidopsis: involvement of phytohormones and transcription factors. Plant Cell Physiol. 56, 98-106 (2015).

[66] Hong, G. J., Xue, X. Y., Mao, Y. B., Wang, L. J. & Chen, X. Y. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell 24, 2635-2648 (2012).

[67] Seo, M. W., Yang, D. S., Kays, S. J., Lee, G. P. & Park, K. W. Sesquiterpene lactones and bitterness in Korean leaf lettuce cultivars. HortScience 44, 246-249 (2009).

[68] Foster, J. G. et al. Influence of cultivation site on sesquiterpene lactone composition of forage Chicory (Cichorium intybus L.). J. Agric. Food Chem. 54, 1772-1778 (2006).