Can transcriptomics shed light on the “old - vine” character of wines? Johan Burger Beatrix Coetzee, Kristin Oosthuizen, Hano Maree
Introduction • Vineyards remain profitable despite prolonged exposure to environmental stresses • Anecdotal evidence - old vines produce wines of greater complexity and depth compared to young vines • ‘Old vine’ on wine labels Old vs. Young vines? • Geographical definitions • South African vines: → ‘old’ > 35 years → ‘young’ < 10 years WineLand · April 2013
Difference in wine quality? GENETICS Viromes & Microbiomes POTENTIAL Epigenetics FACTORS Winemaking Terroir Viticulture
Difference in wine quality? VIROMES Genetics & MICROBIOMES POTENTIAL Epigenetics FACTORS Winemaking Terroir Viticulture
Materials & Methods • Pinotage • (Pinot noir x Cinsaut - 1925) • 8% of total plantings in SA • Genome sequence (Poster 16) • Genome editing (Poster 12) • Samples from a commercial Pinotage young and old inter- planted vineyard in Stellenbosch • Berry and leaf material sampled from nine young and nine old vines at harvest time in Jan 2016 • RNA from 18 vines separately extracted, pooled to yield three young and three old plant biological replicates • Sequencing library prep and RNA- Seq on an Illumina platform was outsourced to a commercial service provider
Sugar & Acid concentration in grape juice Sugar ( o Brix) Acid (g/L) Young 3 Old 3 Young 1 Young 2 Old 1 Old 2 • Juice from 18 vines pooled the same as for RNA • Titratable acid and sugar measured • Young: sugar 22.33°Brix and acid 4.24g/L • Old: sugar 20.4°Brix and acid 5.48g/L
Genes expressed in leaves & berries • Pinot noir has 31 845 Leaves annotated genes 6 902 • 16 027 of these are expressed in Pinotage Not expressed Both tissues • 598 more genes 15 818 2 821 expressed in leaves than in berries Berries 6 304 Loci in V2.1 CRIBI annotation (FPKM ≥ 10)
Differentially Expressed Genes (DEGs) • Of the 16 027 total genes expressed in berries and leaves, 956 genes were differentially expressed between young and old vines. • Five DEGs were present in both berries and leaves.
What are these DEGs? • Functional bins are an indication of roles in metabolism • DEGs involved in fruit ripening as an indicator of wine quality • Focus on genes involved in “ripening” hormone signalling and biochemical changes associated with berry ripening
Berry ripening Vé raison Ripe Fruit set Phase� 1� Phase� 2� Phase� 3� Number� of� differen ally� expressed� genes� Berry� forma on� Berry� ripening� Cell expansion Cell division Cell expansion • “ripening” hormones 4� 1� • 46 DEGs involved in Auxin 8� 2� hormone metabolism and Concentra ons� 5� 1� Ethylene signal transduction 1� 3� identified 1� 0� • Mostly associated with Abscisic acid 1� 1� Hormone� auxin and ethylene Brassinosteriods 0� 5� 0� metabolism 1� 0� 2� Cytokinin 0� 1� Gibberellin 3� 0� 2� 1� Metabolites� Tartaric acid Malic acid Sugars Anthocyanins
Biochemical changes Vé raison Ripe Fruit set Phase� 1� Phase� 2� Phase� 3� Number� of� differen ally� expressed� genes� Berry� forma on� Berry� ripening� Cell expansion Cell division • 203 DEGs Cell expansion involved in 12� 10� biochemical Cell� wall� ripening� 7� 12� changes 5� 0� Sugar� transporters� fruit� 1� 4� during� 11� 5� Secondary� metabolism� 14� 5� changes� 14� 4� Lipid� metabolism� 2� 9� Biochemical� 26� 11� Transporters� 10� 25� 1� 15� Stress� bio c� pathogen� related� proteins� 8� 1�
Sugar & Acid concentration in Grape Juice Sugar ( o Brix) Acid (g/L) Young 1 Young 2 Young 3 Old 1 Old 2 Old 3 • Data suggest that young vines have progressed further along the ripening pathway.
Difference in wine quality? VIROMES Genetics & MICROBIOMES POTENTIAL Epigenetics FACTORS Winemaking Terroir Viticulture
Introduction • 70+ grapevine-infecting virus- & virus-like agents – 65 viruses – GLRaV-3 is the most important • Fungal & bacterial pathogens and endophytes Root microbiome (Hirsch & Mauchline, 2012)
Materials & Methods Nucleic acid extraction Next-generation sequencing Bioinformatic analyses 4 old & 4 young Pinotage vines
Results - Viral diversity HSVd 16000.00 GYSVd-2 Pospiviroidae 14000.00 GYSVd-1 Virus or Viroid Read Ratio* AGVd 12000.00 GSyV-1 10000.00 GRVFV Tymoviridae GFkV 8000.00 GVE 6000.00 GVB Betaflexiviridae GVA 4000.00 GRSPaV 2000.00 GLRaV-2 Closteroviridae GLRaV-3 0.00 Old vines Young vines *Virus or Viroid Read Ratio = read count [contigs of species] / reference genome length * read count [total assembled contigs] * 1E+03 * 1E+06
Results and Discussion – Viral diversity • Greater viral diversity in old vines: → 6 - 8 viruses across old vines (27 viruses, 31 variants detected) → 2 - 5 viruses across young vines (15 viruses, 16 variants detected) • Possible divergent variant of GLRaV-3 • GSyV-1 – not previously detected in South African vines – confirmed by RT-PCR & Sanger sequencing • Five mycoviral families detected – Chrysoviridae , Endornaviridae , Narnaviridae , Partitiviridae and Totiviridae • A contig detected that aligned to a grapevine hammerhead viroid-like RNA sequence – unproven viroid nature (viroid-like RNA entity) • Results confirmed by RT-PCR detection assays
Fungal Diversity 100% Acremonium alternatum 100% Alternaria alternata Alternaria infectoria 90% 90% Alternaria planifunda 80% Amphisphaeriaceae * Relative abundance (%) 80% Ascomycota * Relative abundance 70% Aureobasidium pullulans 70% 60% Cladosporium exasperatum Coniothyrium * 50% 60% Cytospora austromontana 40% Cytospora diatrypelloidea Cytospora magnoliae 50% 30% Diplodia pseudoseriata Discostroma * 20% 40% Dothideomycetes * 10% Epicoccum nigrum 30% Fungi * 0% Lophiostoma winteri 20% Neofusicoccum australe Paraconiothyrium africanum Paraconiothyrium * 10% Samples Pestalotiopsis * Spencermartinsia citricola 0% Sporobolomyces salicinus Old vines Young vines Stemphylium herbarum Tetracladium marchalianum Xylariales * Relative abundance of fungal species ≥ 1% in at least one of eight samples *Unidentified within taxonomic group
Conclusion • First molecular genetics look into the unique character of old Pinotage vines – Suggest that berries of old vines take longer to ripen – More diverse virus community in old vines – Greater fungal diversity in young vines • Can gene expression shed light on the “old - vine” character of wines? – No specific trends observed between young & old vines
So, what next? Genetics Viruses & Microbes POTENTIAL EPIGENETICS FACTORS Winemaking Terroir Viticulture
Acknowledgements
Recommend
More recommend
