Can transcriptomics shed light on the old - vine character of wines? - - PowerPoint PPT Presentation

Can transcriptomics shed light on the “old-vine” character of wines?

Johan Burger

Beatrix Coetzee, Kristin Oosthuizen, Hano Maree

• 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

Introduction

POTENTIAL FACTORS

GENETICS

Epigenetics Terroir Viticulture Winemaking Viromes & Microbiomes

Difference in wine quality?

POTENTIAL FACTORS

Genetics Epigenetics Terroir Viticulture Winemaking

VIROMES & MICROBIOMES

Difference in wine quality?

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

• utsourced to a commercial service

provider

Sugar & Acid concentration in grape juice

Acid (g/L) Sugar (oBrix) Young 1 Young 2 Young 3 Old 1 Old 2 Old 3

• 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

Loci in V2.1 CRIBI annotation (FPKM ≥ 10) Not expressed 15 818 Leaves 6 902 Berries 6 304 Both tissues 2 821

• Pinot noir has 31 845

annotated genes

• 16 027 of these are

expressed in Pinotage

• 598 more genes

expressed in leaves than in berries

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

• f wine quality
• Focus on genes involved

in “ripening” hormone signalling and biochemical changes associated with berry ripening

Fruit set Véraison Ripe

Phase 1 Phase 2 Phase 3 Berry forma on Berry ripening Auxin Sugars Anthocyanins Cytokinin Gibberellin Ethylene Abscisic acid Brassinosteriods

1 4 2 8 1 5 3 1 2 1 1 1 1 3 1 2 5 1

Metabolites Hormone Concentra ons

Number

• f

differen ally expressed genes

Cell division Cell expansion Cell expansion

Malic acid Tartaric acid

Berry ripening

• “ripening” hormones
• 46 DEGs involved in

hormone metabolism and signal transduction identified

• Mostly associated with

auxin and ethylene metabolism

Cell wall Sugar transporters Secondary metabolism Lipid metabolism Transporters Stress bio c pathogen related proteins

12 10 7 12 5 4 1 5 11 5 14 4 14 2 9 11 26 10 25 1 15 1 8

Biochemical changes during fruit ripening

Fruit set Véraison Ripe

Phase 1 Phase 2 Phase 3 Berry forma on Berry ripening

Number

• f

differen ally expressed genes

Cell division Cell expansion Cell expansion

• 203 DEGs

involved in biochemical changes

Biochemical changes

Sugar & Acid concentration in Grape Juice

Acid (g/L) Sugar (oBrix) Young 1 Young 2 Young 3 Old 1 Old 2 Old 3

• Data suggest that young vines have progressed

further along the ripening pathway.

POTENTIAL FACTORS

Genetics Epigenetics Terroir Viticulture Winemaking

VIROMES & MICROBIOMES

Difference in wine quality?

• 70+ grapevine-infecting virus-

& virus-like agents

– 65 viruses – GLRaV-3 is the most important

• Fungal & bacterial pathogens

and endophytes

Introduction

Root microbiome (Hirsch & Mauchline, 2012)

4 old & 4 young Pinotage vines Nucleic acid extraction Next-generation sequencing Bioinformatic analyses

Materials & Methods

Closteroviridae Betaflexiviridae Tymoviridae Pospiviroidae

0.00 2000.00 4000.00 6000.00 8000.00 10000.00 12000.00 14000.00 16000.00

Virus or Viroid Read Ratio* HSVd GYSVd-2 GYSVd-1 AGVd GSyV-1 GRVFV GFkV GVE GVB GVA GRSPaV GLRaV-2 GLRaV-3 Old vines Young vines

Results - Viral diversity

*Virus or Viroid Read Ratio = read count [contigs of species] / reference genome length * read count [total assembled contigs] * 1E+03 * 1E+06

• 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

Results and Discussion – Viral diversity

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Relative abundance (%) Samples Acremonium alternatum Alternaria alternata Alternaria infectoria Alternaria planifunda Amphisphaeriaceae * Ascomycota * Aureobasidium pullulans Cladosporium exasperatum Coniothyrium * Cytospora austromontana Cytospora diatrypelloidea Cytospora magnoliae Diplodia pseudoseriata Discostroma * Dothideomycetes * Epicoccum nigrum Fungi * Lophiostoma winteri Neofusicoccum australe Paraconiothyrium africanum Paraconiothyrium * Pestalotiopsis * Spencermartinsia citricola Sporobolomyces salicinus Stemphylium herbarum Tetracladium marchalianum Xylariales *

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Relative abundance Old vines Young vines Relative abundance of fungal species ≥1% in at least one of eight samples *Unidentified within taxonomic group

Fungal Diversity

• 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

Conclusion

POTENTIAL FACTORS

EPIGENETICS

Genetics Terroir Viticulture Winemaking Viruses & Microbes

So, what next?

Acknowledgements