The absence of the caffeine synthase gene is involved in the naturally decaffeinated status of Coffea humblotiana, a wild species from Comoro archipelago.

Nathalie Raharimalala, Stephane Rombauts, Andrew McCarthy, Andréa Garavito, Simon Orozco-Arias, Laurence Bellanger, Alexa Yadira Morales-Correa, Solène Froger, Stéphane Michaux, Victoria Berry, Sylviane Metairon, Coralie Fournier, Maud Lepelley, Lukas Mueller, Emmanuel Couturon, Perla Hamon, Jean-Jacques Rakotomalala, Patrick Descombes, Romain Guyot, Dominique Crouzillat
Author Information
  1. Nathalie Raharimalala: Centre National de Recherche Appliquée au Développement Rural, BP 1444, 101, Ambatobe, Antananarivo, Madagascar.
  2. Stephane Rombauts: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
  3. Andrew McCarthy: European Molecular Biology Laboratory, 71 Avenue des Martyrs, CS 90181, 38042, Grenoble Cedex 9, France.
  4. Andréa Garavito: Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia.
  5. Simon Orozco-Arias: Department of Systems and Informatics, Universidad de Caldas, Manizales, Colombia.
  6. Laurence Bellanger: Nestle Research-Plant Science Research Unit, BP 49716, 37097, Tours Cedex 2, France.
  7. Alexa Yadira Morales-Correa: Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia.
  8. Solène Froger: Nestle Research-Plant Science Research Unit, BP 49716, 37097, Tours Cedex 2, France.
  9. Stéphane Michaux: Nestle Research-Plant Science Research Unit, BP 49716, 37097, Tours Cedex 2, France.
  10. Victoria Berry: Nestle Research-Plant Science Research Unit, BP 49716, 37097, Tours Cedex 2, France.
  11. Sylviane Metairon: Nestle Research, Société des Produits Nestlé SA, 1015, Lausanne, Switzerland.
  12. Coralie Fournier: Nestle Research, Société des Produits Nestlé SA, 1015, Lausanne, Switzerland.
  13. Maud Lepelley: Nestle Research-Plant Science Research Unit, BP 49716, 37097, Tours Cedex 2, France.
  14. Lukas Mueller: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, 14853, USA.
  15. Emmanuel Couturon: Institut de Recherche pour le Développement, UMR DIADE, Université de Montpellier, Montpellier, France.
  16. Perla Hamon: Institut de Recherche pour le Développement, UMR DIADE, Université de Montpellier, Montpellier, France.
  17. Jean-Jacques Rakotomalala: Centre National de Recherche Appliquée au Développement Rural, BP 1444, 101, Ambatobe, Antananarivo, Madagascar.
  18. Patrick Descombes: Nestle Research, Société des Produits Nestlé SA, 1015, Lausanne, Switzerland.
  19. Romain Guyot: Universidad Autónoma de Manizales, Manizales, Colombia. romain.guyot@ird.fr.
  20. Dominique Crouzillat: Nestle Research-Plant Science Research Unit, BP 49716, 37097, Tours Cedex 2, France. dominique.crouzillat@rdto.nestle.com.

Abstract

Caffeine is the most consumed alkaloid stimulant in the world. It is synthesized through the activity of three known N-methyltransferase proteins. Here we are reporting on the 422-Mb chromosome-level assembly of the Coffea humblotiana genome, a wild and endangered, naturally caffeine-free, species from the Comoro archipelago. We predicted 32,874 genes and anchored 88.7% of the sequence onto the 11 chromosomes. Comparative analyses with the African Robusta coffee genome (C. canephora) revealed an extensive genome conservation, despite an estimated 11 million years of divergence and a broad diversity of genome sizes within the Coffea genus. In this genome, the absence of caffeine is likely due to the absence of the caffeine synthase gene which converts theobromine into caffeine through an illegitimate recombination mechanism. These findings pave the way for further characterization of caffeine-free species in the Coffea genus and will guide research towards naturally-decaffeinated coffee drinks for consumers.

References

  1. Charrier, A. Rapport de Mission aux Comores. 1–8 (1971).
  2. International Union for Conservation of Nature (IUCN). The IUCN Red List of Threatened Species (2020).
  3. Davis, A. P. et al. High extinction risk for wild coffee species and implications for coffee sector sustainability. Sci. Adv. 5, eaav3473 (2019). pubmed:30746478; pmcid:6357749; doi:10.1126/sciadv.aav3473
  4. Chadburn, H. & Davis, A. P. Coffea humblotiana, Caféier de Humblot. The IUCN Red List of Threatened Species 2017 e.T108652718A108665565 (2017).
  5. Guyot, R. et al. WCSdb: A database of Wild Coffea Species. Database (2020).
  6. WCSP. World Checklist of Selected Plant Families (Royal Botanic Gardens, 2018).
  7. Hamon, P. et al. Genotyping-by-sequencing provides the first well-resolved phylogeny for coffee (Coffea) and insights into the evolution of caffeine content in its species: GBS coffee phylogeny and the evolution of caffeine content. Mol. Phylogenet. Evol. 109, 20 (2017). doi:10.1016/j.ympev.2017.02.009
  8. Yu, Q. et al. Micro-collinearity and genome evolution in the vicinity of an ethylene receptor gene of cultivated diploid and allotetraploid coffee species (Coffea): Recent speciation event of Coffea arabica. Plant J. 67, 305–317 (2011). pubmed:21457367; doi:10.1111/j.1365-313X.2011.04590.x
  9. Bertrand,. Sur la Composition Biochimique du Café de la Grande Comore 162–164 (G. Masson, 1901).
  10. Campa, C., Doulbeau, S., Dussert, S., Hamon, S. & Noirot, M. Diversity in bean Caffeine content among wild Coffea species: Evidence of a discontinuous distribution. Food Chem. 91, 633–637 (2005). doi:10.1016/j.foodchem.2004.06.032
  11. Ashihara, H. Metabolism of alkaloids in coffee plants. Braz. J. Plant Physiol. 18, 1–8 (2006). doi:10.1590/S1677-04202006000100001
  12. Ky, C. L. et al. Caffeine, trigonelline, chlorogenic acids and sucrose diversity in wild Coffea arabica L. and C. canephora P accessions. Food Chem. 75, 223–230 (2001). doi:10.1016/S0308-8146(01)00204-7
  13. Perrois, C. et al. Differential regulation of caffeine metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta). Planta 241, 179–191 (2014). pubmed:25249475; pmcid:4282694; doi:10.1007/s00425-014-2170-7
  14. Ashihara, H., Sano, H. & Crozier, A. Caffeine and related purine alkaloids: Biosynthesis, catabolism, function and genetic engineering. Phytochemistry 69, 841–856 (2008). pubmed:18068204; doi:10.1016/j.phytochem.2007.10.029
  15. Denoeud, F. et al. The coffee genome provides insight into the convergent evolution of caffeine biosynthesis. Science 345, 1181–1184 (2014). pubmed:25190796; doi:10.1126/science.1255274
  16. Lang, T. et al. Numerous compounds orchestrate coffee’s bitterness. J. Agric. Food Chem. 68, 6692–6700 (2020). pubmed:32437139; doi:10.1021/acs.jafc.0c01373
  17. Tran, H. T., Lee, L. S., Furtado, A., Smyth, H. & Henry, R. J. Advances in genomics for the improvement of quality in coffee. J. Sci. Food Agric. 96, 3300–3312 (2016). pubmed:26919810; doi:10.1002/jsfa.7692
  18. Leloup, V., Louvrier, A. & Liardon, R. Degradation Mechanisms of Chlorogenic Acids During Roasting. 192–198 (1995).
  19. Ogita, S., Uefuji, H., Yamaguchi, Y., Koizumi, N. & Sano, H. Producing decaffeinated coffee plants. Nature 423, 823–823 (2003). pubmed:12815419; doi:10.1038/423823a
  20. Charrier, A. La structure genetique des cafeiers spontanes de la region Malgache et leurs relations avec les cafeiers d’arigine Africaine (Eucoffea). (1978).
  21. Mazzafera, P. & Carvalho, A. Breeding for low seed caffeine content of coffee (Coffea L.) by interspecific hybridization. Euphytica 59, 55–60 (1991). doi:10.1007/BF00025361
  22. Silvarolla, M. B., Mazzafera, P. & Fazuoli, L. C. A naturally decaffeinated arabica coffee. Nature 429, 826 (2004). pubmed:15215853; doi:10.1038/429826a
  23. Maluf, M. P. et al. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica. Genet. Mol. Biol. 32, 802–810 (2009). pubmed:21637458; pmcid:3036905; doi:10.1590/S1415-47572009005000090
  24. Mazzafera, P., Baumann, T. W., Shimizu, M. M. & Silvarolla, M. B. Decaf and the steeplechase towards decaffito—the coffee from caffeine-free Arabica plants. Trop. Plant Biol. 2, 63–76 (2009). doi:10.1007/s12042-009-9032-7
  25. Razafinarivo, N. J. et al. Genetic structure and diversity of coffee (Coffea) across Africa and the Indian Ocean islands revealed using microsatellites. Ann. Bot. 111, 20 (2013). doi:10.1093/aob/mcs283
  26. Ou, S., Chen, J. & Jiang, N. Assessing genome assembly quality using the LTR Assembly Index (LAI). Nucleic Acids Res. https://doi.org/10.1093/nar/gky730 (2018). doi:10.1093/nar/gky730; pubmed:30107434; pmcid:6323972
  27. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012). pubmed:22388286; pmcid:3322381; doi:10.1038/nmeth.1923
  28. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019). pubmed:31375807; pmcid:7605509; doi:10.1038/s41587-019-0201-4
  29. Ma, J. & Bennetzen, J. L. Rapid recent growth and divergence of rice nuclear genomes. Proc. Natl. Acad. Sci. 101, 12404–12410 (2004). pubmed:15240870; doi:10.1073/pnas.0403715101; pmcid:515075
  30. Proost, S. et al. i-ADHoRe 3.0—fast and sensitive detection of genomic homology in extremely large data sets. Nucleic Acids Res. 40, e11–e11 (2012). pubmed:22102584; doi:10.1093/nar/gkr955
  31. Cabanettes, F. & Klopp, C. D-GENIES: Dot plot large genomes in an interactive, efficient and simple way. PeerJ 6, e4958 (2018). pubmed:29888139; pmcid:5991294; doi:10.7717/peerj.4958
  32. Ribas, A. F., Cenci, A., Combes, M. C., Etienne, H. & Lashermes, P. Organization and molecular evolution of a disease-resistance gene cluster in coffee trees. BMC Genom. 12, 20 (2011). doi:10.1186/1471-2164-12-240
  33. Xu, Z. et al. Tandem gene duplications drive divergent evolution of caffeine and crocin biosynthetic pathways in plants. BMC Biol. 18, 1–14 (2020). doi:10.1186/s12915-020-00795-3
  34. Ly, S. N. et al. Chloroplast genomes of Rubiaceae: Comparative genomics and molecular phylogeny in subfamily Ixoroideae. PLoS One 15, e0232295 (2020). pubmed:32353023; pmcid:7192488; doi:10.1371/journal.pone.0232295
  35. Wicker, T., Yahiaoui, N. & Keller, B. Illegitimate recombination is a major evolutionary mechanism for initiating size variation in plant resistance genes. Plant J. 51, 631–641 (2007). pubmed:17573804; doi:10.1111/j.1365-313X.2007.03164.x
  36. Davis, A. P. et al. A global assessment of distribution, diversity, endemism, and taxonomic effort in the Rubiaceae. Ann. Missouri Bot. Garden 20, 68–78 (2009). doi:10.3417/2006205
  37. Wendel, J. F., Jackson, S. A., Meyers, B. C. & Wing, R. A. Evolution of plant genome architecture. Genome Biol. 17, 37 (2016). pubmed:26926526; pmcid:4772531; doi:10.1186/s13059-016-0908-1
  38. Guyot, R. et al. Ancestral synteny shared between distantly-related plant species from the asterid (Coffea canephora and Solanum Sp.) and rosid (Vitis vinifera) clades. BMC Genom. 13, 103 (2012). doi:10.1186/1471-2164-13-103
  39. Razafinarivo, N. J. et al. Geographical gradients in the genome size variation of wild coffee trees (Coffea) native to Africa and Indian Ocean islands. Tree Genet. Genomes 8, 1345–1358 (2012). doi:10.1007/s11295-012-0520-9
  40. Guyot, R. et al. Partial sequencing reveals the transposable element composition of Coffea genomes and provides evidence for distinct evolutionary stories. Mol. Genet. Genom. 291, 1979–1990 (2016). doi:10.1007/s00438-016-1235-7
  41. Jin, J.-Q. et al. Hongyacha, a naturally caffeine-free tea plant from Fujian, China. J. Agric. Food Chem. 66, 11311–11319 (2018). pubmed:30303011; doi:10.1021/acs.jafc.8b03433
  42. Mizuno, K. et al. Isolation of a new dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.). FEBS Lett. 534, 75–81 (2003). pubmed:12527364; doi:10.1016/S0014-5793(02)03781-X
  43. Deng, C. et al. Metabolite and transcriptome profiling on xanthine alkaloids-fed tea plant (Camellia sinensis) shoot tips and roots reveal the Complex metabolic network for caffeine biosynthesis and degradation. Front. Plant Sci. 11, 551288 (2020). pubmed:33013969; pmcid:7509060; doi:10.3389/fpls.2020.551288
  44. Deng, W. W., Rakotomalala, J.-J., Nagai, C. & Ashihara, H. Caffeine biosynthesis and purine metabolism in leaves of mascarocoffea species. Eur. Chem. Bull. 6, 223 (2017). doi:10.17628/ecb.2017.6.223-228
  45. Ashihara, H. et al. Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes. Phytochemistry 67, 882–886 (2006). pubmed:16624354; doi:10.1016/j.phytochem.2006.02.016
  46. Davis, A. P., Tosh, J., Ruch, N. & Fay, M. F. Growing coffee: Psilanthus (Rubiaceae) subsumed on the basis of molecular and morphological data; implications for the size, morphology, distribution and evolutionary history of Coffea: Psilanthus subsumed in Coffea. Bot. J. Linn. Soc. 167, 357–377 (2011). doi:10.1111/j.1095-8339.2011.01177.x
  47. Hamon, P. et al. Caffeine-free species in the genus coffea. Coffee Health Dis. Prev. 20, 39–44. https://doi.org/10.1016/B978-0-12-409517-5.00005-X (2015). doi:10.1016/B978-0-12-409517-5.00005-X
  48. Clifford, M. N., Gibson, C. L., Rakotomalala, J.-J.R., Cros, E. & Charrier, A. Caffeine from green beans of Mascarocoffea. Phytochemistry 30, 4039–4040 (1991). doi:10.1016/0031-9422(91)83461-S
  49. Michelmore, R. W. & Meyers, B. C. Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res. 8, 1113–1130 (1998). pubmed:9847076; doi:10.1101/gr.8.11.1113
  50. Chevalier, A. Un nouveau Caféier sauvage de Madagascar à grains sans caféine. Rev. Bot. Appl. Agric. Colon. 17, 821–826 (1937).
  51. Dornano, M., Chassevent, F. & Pougneaud, S. Composition et caractéristiques chimiques de Coffea sauvages de Madagascar. II. Recherche de la caféine et d’autres méthylxanthines dans les feuilles et les graines de caféiers sauvages et cultivés. III. Cafamarine et trigonelline contenues dans les graines de trois caféiers sauvages. Café Cacao 11, 235–249 (1967).
  52. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). pubmed:24695404; pmcid:4103590; doi:10.1093/bioinformatics/btu170
  53. Marçais, G. & Kingsford, C. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics 27, 764–770 (2011). pubmed:21217122; pmcid:3051319; doi:10.1093/bioinformatics/btr011
  54. Chin, C.-S. et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 13, 1050–1054 (2016). pubmed:27749838; pmcid:5503144; doi:10.1038/nmeth.4035
  55. English, A. C. et al. Mind the Gap: Upgrading genomes with Pacific biosciences RS long-read sequencing technology. PLoS One 7, e47768 (2012). pubmed:23185243; pmcid:3504050; doi:10.1371/journal.pone.0047768
  56. Tarailo-Graovac, M. & Chen, N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinform. 25, 20 (2009). doi:10.1002/0471250953.bi0410s25
  57. Nachtweide, S. & Stanke, M. Multi-genome annotation with AUGUSTUS. In Gene Prediction, vol 1962 (ed. Kollmar, M.) 139–160 (Springer, 2019).
  58. Gremme, G., Brendel, V., Sparks, M. E. & Kurtz, S. Engineering a software tool for gene structure prediction in higher organisms. Inf. Softw. Technol. 47, 965–978 (2005). doi:10.1016/j.infsof.2005.09.005
  59. Haas, B. J. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol. 9, R7 (2008). pubmed:18190707; pmcid:2395244; doi:10.1186/gb-2008-9-1-r7
  60. Emms, D. M. & Kelly, S. OrthoFinder: Phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019). pubmed:31727128; pmcid:6857279; doi:10.1186/s13059-019-1832-y
  61. McCarthy, E. M. & McDonald, J. F. LTR_STRUC: A novel search and identification program for LTR retrotransposons. Bioinformatics 19, 362–367 (2003). pubmed:12584121; doi:10.1093/bioinformatics/btf878
  62. Orozco-Arias, S. et al. Inpactor, integrated and parallel analyzer and classifier of LTR retrotransposons and its application for pineapple LTR retrotransposons diversity and dynamics. Biology 7, 32 (2018). pmcid:6022998; doi:10.3390/biology7020032
  63. Yu, Y., Ouyang, Y. & Yao, W. shinyCircos: An R/Shiny application for interactive creation of Circos plot. Bioinformatics 34, 1229–1231 (2018). pubmed:29186362; doi:10.1093/bioinformatics/btx763
  64. Ming, R. et al. The pineapple genome and the evolution of CAM photosynthesis. Nat. Genet. 47, 1435–1442 (2015). pubmed:26523774; pmcid:4867222; doi:10.1038/ng.3435
  65. Sonnhammer, E. L. L. & Durbin, R. A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 167, GC1–GC10 (1995). pubmed:8566757; doi:10.1016/0378-1119(95)00714-8
  66. Rice, P., Longden, I. & Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000). pubmed:10827456; doi:10.1016/S0168-9525(00)02024-2
  67. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990). doi:10.1016/S0022-2836(05)80360-2; pubmed:2231712
  68. Quinlan, A. R. & Hall, I. M. BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010). pubmed:20110278; pmcid:2832824; doi:10.1093/bioinformatics/btq033
  69. Contreras-Moreira, B. et al. Analysis of plant pan-genomes and transcriptomes with GET_HOMOLOGUES-EST, a clustering solution for sequences of the same species. Front. Plant Sci. 8, 20 (2017). doi:10.3389/fpls.2017.00184
  70. Sievers, F. & Higgins, D. G. Clustal omega. Curr. Protoc. Bioinform. 48, 20 (2014). doi:10.1002/0471250953.bi0313s48
  71. Gouy, M., Guindon, S. & Gascuel, O. SeaView Version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27, 221–224 (2010). pubmed:19854763; doi:10.1093/molbev/msp259
  72. Gouet, P., Robert, X. & Courcelle, E. ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 31, 3320–3323 (2003). pubmed:12824317; pmcid:168963; doi:10.1093/nar/gkg556

MeSH Term

Amino Acid Sequence
Caffeine
Chromosomes, Plant
Coffea
Comoros
Comparative Genomic Hybridization
Evolution, Molecular
Methyltransferases
Phylogeny
Plant Leaves
Plant Proteins
Sequence Alignment
Sequence Analysis, RNA
Theobromine

Chemicals

Plant Proteins
Caffeine
Methyltransferases
caffeine synthase
Theobromine

Word Cloud