A key ‘foxy’ aroma gene is regulated by homology-induced promoter indels in the iconic juice grape ‘Concord’

Abstract

‘Concord’, the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special ‘foxy’ aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key ‘foxy’ aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in ‘Concord’ and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the ‘foxy’ aroma trait and makes it genetically trackable and amenable in grapevine breeding.

References

[1] Fortes, A. M. & Pais, M. S. in Nutritional Composition of Fruit Cultivars Ch. 12, 257-286 (2016). https://doi.org/10.1016/B978-0-12-408117-8.00012-X.

[2] Eibach, R. & Töpfer, R. in Grapevine Breeding Programs for the Wine Industry 1-22 (2015). https://doi.org/10.1016/B978-1-78242-075-0.00001-6.

[3] Schofield, E. A. ‘He Sowed; Others Reaped’: Ephraim Wales Bull and the Origins of the “Concord” Grape. Arnoldia 48, 4-15 (1988).

[4] Sawler, J. et al. Genomics assisted ancestry deconvolution in grape. PLoS ONE 8, 1-8 (2013).

[5] Huber, F. et al. A view into American grapevine history: Vitis vinifera cv. ‘Sémillon’ is an ancestor of ‘Catawba’ and ‘Concord’. Vitis-J. Grapevine Res. 55, 53-56 (2016).

[6] Morris, J. R. & Striegler, K. in Processing Fruits: Science and Technology, Vol. 2: Major Processed Products (eds. Somogyi, L. P., Barrett, D. M. & Hui, Y. H.) 197-234 (Lancater: Technomic Publishing Company, Lancaster, 1996).

[7] Rowe, C. A., Nantz, M. P., Nieves, C., West, R. L. & Percival, S. S. Regular consumption of concord grape juice benefits human immunity. J. Med. Food 14, 69-78 (2011).

[8] Shure, K. B. & Acree, T. E. in Fruit Flavor: Biogenesis, Characterization, and Authentication (eds. Rouseff, R. L. & Leahy, M. M.) 127-133 (American Chemical Society, 1995).

[9] Sun, Q., Gates, M. J., Lavin, E. H., Acree, T. E. & Sacks, G. L. Comparison of odor-active compounds in grapes and wines from vitis vinifera and non-foxy American grape species. J. Agric. Food Chem. 59, 10657-10664 (2011).

[10] González-Barreiro, C., Rial-Otero, R., Cancho-Grande, B. & Simal-Gándara, J. Wine aroma compounds in grapes: a critical review. Crit. Rev. Food Sci. Nutr. 55, 202-218 (2015).

[11] Power, F. B. & Chesnut, V. K. Examination of authentic grape juices for methyl anthranilate. J. Agric. Res. XXIII, 47-53 (1923).

[12] Acree, T. E., Lavin, E. H., Nishida, R. & Watanabe, S. O-Amino acetophenone the ‘foxy’ smelling component of labruscana grapes. In Flavour Science and Technology-6th Weurmann Symposium (eds. Bessiere, Y. & Thomas, A. F.) 49-52 (Wiley-Blackwell, 1990).

[13] Wang, J. & Luca, V. De . The biosynthesis and regulation of biosynthesis of Concord grape fruit esters, including ‘foxy’ methylanthranilate. Plant J. 44, 606-619 (2005).

[14] Slegers, A., Angers, P., Ouellet, É., Truchon, T. & Pedneault, K. Volatile compounds from grape skin, juice and wine from five interspecific hybrid grape cultivars grown in Québec (Canada) for wine production. Molecules 20, 10980-11016 (2015).

[15] Prudêncio Dutra, M. et al. Rapid determination of the aromatic compounds methyl-anthranilate, 2′-aminoacetophenone and furaneol by GC-MS: Method validation and characterization of grape derivatives. Food Res. Int. 107, 613-618 (2018).

[16] Pillet, J. et al. Identification of a methyltransferase catalyzing the final step of methyl anthranilate synthesis in cultivated strawberry. BMC Plant Biol. 17, 1-12 (2017).

[17] Kollner, T. G. et al. Herbivore-Induced SABATH methyltransferases of maize that methylate anthranilic acid using S-adenosyl-L-methionine. Plant Physiol. 153, 1795-1807 (2010).

[18] Jaillon, O. et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449, 463-467 (2007).

[19] Velasco, R. et al. A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. PLoS ONE 2, e1236 (2007).

[20] Tabidze, V. et al. Whole genome comparative analysis of four Georgian grape cultivars. Mol. Genet. Genomics 292, 1377-1389 (2017).

[21] Da Silva, C. et al. The high polyphenol content of grapevine cultivar tannat berries is conferred primarily by genes that are not shared with the reference genome. Plant Cell 25, 4777-4788 (2013).

[22] Girollet, N., Rubio, B. & Bert, P.-F. De novo phased assembly of the Vitis riparia grape genome. bioRxiv https://doi.org/10.1101/640565 (2019).

[23] Canaguier, A. et al. A new version of the grapevine reference genome assembly (12X.v2) and of its annotation (VCost.v3). Genomics Data 14, 56-62 (2017).

[24] Di Genova, A. et al. Whole genome comparison between table and wine grapes reveals a comprehensive catalog of structural variants. BMC Plant Biol. 14, 7 (2014).

[25] Patel, S. et al. Comparison of three assembly strategies for a heterozygous seedless grapevine genome assembly. BMC Genomics 19, 57 (2018).

[26] Coetzee, B. Genome and Transcriptome Sequencing of Vitis vinifera cv Pinotage Phd thesis, Stellenbosch University (2018).

[27] Gambino, G. et al. Whole-genome sequencing and SNV genotyping of ‘Nebbiolo’ (Vitis vinifera L.) clones. Sci. Rep. 7, 17294 (2017).

[28] Mercenaro, L., Nieddu, G., Porceddu, A., Pezzotti, M. & Camiolo, S. Sequence polymorphisms and structural variations among four grapevine (Vitis vinifera L.) cultivars representing Sardinian agriculture. Front. Plant Sci. 8, 1279 (2017).

[29] Chin, C. S. et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 13, 1050-1054 (2016).

[30] Roach, M. J. et al. Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay. PLoS Genet. 14, 1-24 (2018).

[31] Zhou, Y. S. et al. Structural variants, clonal propagation, and genome evolution in grapevine (Vitis vinifera). bioRxiv https://doi.org/10.1101/508119 (2018).

[32] Vondras, A. M., Minio, A., Blanco-ulate, B. & Figueroa-balderas, R. The genomic diversification of clonally propagated grapevines. bioRxiv https://doi.org/10.1101/585869 (2019).

[33] Minio, A., Massonnet, M., Figueroa-Balderas, R., Castro, A. & Cantu, D. Diploid genome assembly of the wine grape Carménère. Genes Genomes Genet. 9, 1331-1337 (2019).

[34] Fasoli, M. et al. Pectins, hemicelluloses and celluloses show specific dynamics in the internal and external surfaces of grape berry skin during ripening. Plant Cell Physiol. 57, 1332-1349 (2016).

[35] Hadfield, K. A. & Bennett, A. B. Polygalacturonases: many genes in search of a function. Plant Physiol. 117, 337-343 (2002).

[36] Grncarevic, M. & Radler, F. A review of the surface lipids of grapes and their importance in the drying process. Am. J. Enol. Vitic. 22, 80-86 (1971).

[37] Yamamura, H. & Naito, R. Surface wax of several grapes in Japan. J. Jpn. Soc. Hort. Sci. 52, 266-272 (1983).

[38] Chen, X., Goodwin, S. M., Boroff, V. L., Liu, X. & Jenks, M. A. Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell 15, 1170-1185 (2003).

[39] Ma, Z. Y. et al. Phylogenomics, biogeography, and adaptive radiation of grapes. Mol. Phylogenet. Evol. 129, 258-267 (2018).

[40] Zhou, Y., Massonnet, M., Sanjak, J. S., Cantu, D. & Gaut, B. S. Evolutionary genomics of grape (Vitis vinifera ssp. vinifera) domestication. Proc. Natl Acad. Sci. USA 114, 11715-11720 (2017).

[41] Marroni, F. et al. Reduction of heterozygosity (ROH) as a method to detect mosaic structural variation. Plant Biotechnol. J. 15, 791-793 (2017).

[42] Dal Santo, S. et al. Grapevine field experiments reveal the contribution of genotype, the influence of environment and the effect of their interaction (G×E) on the berry transcriptome. Plant J. 93, 1143-1159 (2018).

[43] Cardone, M. F. et al. Inter-varietal structural variation in grapevine genomes. Plant J. 88, 648-661 (2016).

[44] Liang, Z. et al. Whole-genome resequencing of 472 Vitis accessions for grapevine diversity and demographic history analyses. Nat. Commun. 10, 1-12 (2019).

[45] Kim, H. S. et al. Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis. J. Biol. Chem. 282, 36292-36302 (2007).

[46] Burley, S. K. & Roeder, R. G. Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem. 65, 769-799 (1996).

[47] Witte, C.-P., Le, Q. H., Bureau, T. & Kumar, A. Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes. Proc. Natl Acad. Sci. 98, 13778-13783 (2002).

[48] Nelson, R. R., Acree, T. E. & Butts, R. M. Methyle anthranilate as an aroma constituent of American wine. J. Food Sci. 42, 57-59 (1977).

[49] Braun, F. in Vitis zur Frühdiagnose von Weinqualität. (Justus-Liebig-Universität Gießen, 2017).

[50] Fisher, K. H., Fuleki, T. & Reynolds, A. G. The heritability of methyl anthranilate and total volatile esters in Vitis spp. hybrids. In Vitis (special issue) Proc. 5th Int. Symp. Grape Breeding (eds. Hammerschlag, F. & Litz, R. E.) 159-168 (CAB International, Wallingford, 1990).

[51] Reynolds, A. G., Fuleki, T. & Evans, W. D. Inheritance of methyl anthranilate and total volatile esters in Vitis spp. Am. J. Enol. Vitic. 33, 14-19 (1981).

[52] Leach, D. R. F. Long DNA palindromes, cruciform structures, genetic instability and secondary structure repair. BioEssays 16, 893-900 (1994).

[53] Bzymek, M. & Lovett, S. T. Instability of repetitive DNA sequences: the role of replication in multiple mechanisms. Proc. Natl Acad. Sci. 98, 8319-8325 (2002).

[54] Chen, X. et al. Molecular analysis of a deletion hotspot in the NRXN1 region reveals the involvement of short inverted repeats in deletion CNVs. Am. J. Hum. Genet. 92, 375-386 (2013).

[55] Lee, J. A., Carvalho, C. M. B. & Lupski, J. R. A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 131, 1235-1247 (2007).

[56] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402-408 (2001).

[57] Nakagawa, T. et al. Improved gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095-2100 (2007).

[58] Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743 (1998).