Sustainability in grape production and commerce d Presented by - PowerPoint PPT Presentation

Sustainability in grape production and commerce d • Presented by Vincenzo De Luca Presented by Vincenzo De Luca – Supported by OMAFRA, NSERC and Industry • Dr. Kyung Hee Kim • Brent Wiens, MSc • Dawn Hall, PhD Introduction 1. Research Interests: cell specialization and biochemical production d ti 2. Acyltransferases involved in grape volatile production. • Acyl esters of phenols and terpenes. • Relationship between anthocyanin 5 glucosides and methyl anthranilate anthranilate. 3. Glucosyltransferases and grape natural products. • Water solubility, stability, transport and compartmentation • Bifunctional GT and its role in resveratrol and phenol tartrates production production 4. Identification of Grape Pomace secondary metabolites. • Value as food additives

Sustainability Sustainability • Regulatory issues • Wine grapes – Demands by governments to – Are clonally propagated decrease the pesticide p – Large numbers of identical L b f id ti l footprint for wine grape individuals growing together production. – Susceptible to similar • Contamination of land, water diseases and air. – Unique to wine grapes U i t i • Population risks P l ti i k – Lack of large scale breeding – Needs for biocontrol measures efforts over pesticide use • Unique to wine grapes – Need for genetically superior disease resistant genotypes di i t t t – Lack of large scale breeding • Risks efforts – Heavy dependence on – Appearance of new diseases? management and chemical g • Pierce’s Disease pesticide use • Powdery Mildew • New forms of Phylloxera

Approaches to grape improvement Approaches to grape improvement • Tools available • Examples of important – Genomics wine related pathways • Grape genome sequence (Pinot Noir) sequence (Pinot Noir) – Anthocyanins Anthocyanins • Synteny • Very well known in plants • Marker assisted – Resveratrol breeding breeding • Very well known in grape – Metabolic profiles – Acylated aroma and – Pathways flavor cpds p • Genes G • Methyl anthranilate • Proteins – C-13 Norisoprenoids • Mutations ฀  -damascenone ฀  -damascenone

Cell specific specialization for secondary metabolism O COOH OGlu O CH HC OH HO COOHGluO GH Tryptophan Geraniol R R DS DS OGl u TDC SLS Resveratrol Glucosides Phenol tartrates Tryptamine Secologanin O STR1 Strictosidine OCH 3 OCH 3 SG Ajmalicine NH 2 Desacetoxyvindoline Tabersonine Catharanthine Methyl Anthranilate T16H D4H OH DAT DAT Desacetylvindoline Vindoline N C 2 H 5 N H H CO C H 3 CO 2 C N N H H 3 CO N OAc H CO 2 CH 3 H 3 C HO Catharanthus roseus Catharanthus roseus Anhydrovinblastine Antineoplastic agent Vincenzo De Luca, Brock University, St. Catharines,Ontario, Canada, February 22, 2010

Cell factories & biosynthesis of plant natural products Scanning electron microscopy of aromatic Sage: The Scientist Nov 17 2003 V17: p12 Brock, April 19, 2010

Research Interests of our Laboratory • Biosynthesis of Novel and commercially important metabolites • Evolution of biosynthetic pathways • Cell and biochemical specialization involved 7 Only 25 h required to fill this gland with fill this gland with 1 monoterpenes and 6 2 Mint gland on flavonoids 3 5 4 the surface of a mint leaf mint leaf 8 9 10 CO 2 2 + Sugars photosynthesis light Diagram from: Plant Physiology 136:4215-4227 (2004)

Anthocyanin Biosynthesis is the best know pathway of Secondary metabolism in plants OH 3 malonylCoA HO OH + CHS p-coumarylCoA OH O OH OH CHI OH OH OH HO O HO O HO O OH F3'H F3'5'H OH O OH O OH O F3H OH OH OH OH OH OH OH OH OH OH OH OH HO O HO O HO O HO O HO O OH OH FLS FLS F3'H F3'5'H OH OH OH OH OH OH O OH O OH O OH O OH O DFR DFR DFR DFR FLS OH ANR LDOX ANR LDOX HO O UFGT OR 1 UFGT OR 1 OH OH OH + OH O + HO HO O O HO O O O OR 3 OGlu OGlu OR 2 O OR 2 O

The pattern of grape pigmentation in nine cultivars is regulated by differential gene expression of is regulated by differential gene expression of anthocyanin biosynthesis and their transcription factors Castellarin and Di Gaspero BMC Plant Biology (2007) 7: 46

Anthocyanin Biosynthesis is the best know pathway of Secondary metabolism in plants OH 3 malonylCoA HO OH + CHS p-coumarylCoA OH O OH OH OH OH CHI OH OH OH HO O HO O HO O OH F3'H F3'5'H OH O OH O OH O F3H F3H OH OH OH OH OH OH OH OH OH OH HO O HO O HO O HO O HO O OH OH FLS F3'H FLS F3'5'H OH OH OH OH OH OH O OH O OH O OH O OH O DFR DFR FLS OH ANR LDOX ANR LDOX HO O UFGT OR 1 UFGT OR 1 OH OH OH + OH O + HO HO O O HO O OR 3 OGlu OGlu OR 2 O OR 2 O • The sugar pattern is used to define wines contaminated with North American grapes contaminated with North American grapes. • However anthocyanins with sugars on the 5 position are more stable and their presence could be valuable for wine color stability?

Identification of the 5GT responsible for the f formation of anthocyanin diglucosides ti f th i di l id R 1 R 1 OH OH HO HO O O HO HO O O 5-GT R 2 R 2 UDP-Glucose O OH O OH O O O HO O HO O HO O HO OH OH OR 3 OR 3 OH OH OH OH The inability of most European grapevines to produce • 3,5-di-O-glucosides has long been used to classify wines according to their varietal origin. i di h i i l i i – This study showed that V. vinifera has a 5GT gene with 2 mutations that render the protein inactive. – Correction of the 2 mutations reactivated the V. vinifera 5GT gene This explains why revertants have not been observed – Janvary et al, Agric Food Chemistry (2009) 57: 3512-3518

Colocalization of 5GT and AMAT to the same area of chromosome 9 explains why FOXY & diglucosides are linked! p y g R 1 R 1 OH OH HO O HO O 5-GT R 2 R 2 UDP-Glucose O OH O OH O O O HO O HO O HO O HO OH OR 3 OR 3 OH OH OH OH OH OH 5GT double AMAT-like mutant gene (CAO23156) V vinifera ( Pinot Noir) genome shows that : V. vinifera ( Pinot Noir) genome shows that : • • – CAO23156 is 95% identical on the amino acid level with V. labrusca anthraniloyl-CoA:methanol anthraniloyl transferase (AMAT). – Colocalization of the two genes would explain genetic linkage between these 2 traits in hybrid cultivars. Janvary et al, Agric Food Chemistry (2009) 57: 3512-3518

OH How are floral Lutein HO norisoprenoids made? Cryptoxanthin HO OH Journal of Experimental Botany Vol 56 Journal of Experimental Botany, Vol. 56, No. 420, pp. 2721–2731, October 2005 HO Zeaxanthin VvCCDs O O www.vcbio.science.ru.nl/. HO ../applets/chloroplast/  3-hydroxy- -ionone • Grapes Grapes Oxidases, Reductases & Dehydrogenases Oxidases Reductases & Dehydrogenases – Muscat grapes make more OH OH norisoprenoids than in Muscat of Alexandria grapes than in HO O O those of Shiraz   3-hydroxy- -ionol 3-oxo- -ionol – Si Single VvCCD gene appears to l V CCD t HO be involved 3-hydroxy-7,8- O O – VvCCD molecular marker can be  dihydro- -ionone used for selection purposes in HO breeding program to enhance g p g   3-hydroxy- - 3 hydroxy  -damascenone the norisoprenoid profiles of damascenone wine grapes

How are floral volatile acylated flavor compounds made made? Example: Grapes make methyl anthranilate from pectin derived methanol and Example: Grapes make methyl anthranilate from pectin derived methanol and the CoA ester of anthranilate Plant J the CoA ester of anthranilate Plant J (2005) 44, 606–619 the CoA ester of anthranilate Plant J (2005) 44, 606 619 the CoA ester of anthranilate Plant J OCH 3 H H O HO H O O HO H O OCH 3 O H H O H O OH O HO H H HO H O O H HO O OCH 3 H H H H O Pectin Methylesterase H OH H O HO H O O HO H O OH Ce Cell Wall softening a so e g O H H O H O OH OH O HO H H HO H O O during ripening H HO O OH H H H H 4 92 2 2 O MeOH O OCH 3 O OH + O SCoA NH 2 NH 2 Coenzyme A NH 2 AMAT % distribution of AMAT Ligase In grape Anthranilic acid Methyl anthranilate Anthraniloyl CoA Cross-section Concord wk16

European Grape cultivars have almost identical transcripts to those of AMAT of identical transcripts to those of AMAT of Concord Grape? Different varieties of mature Vitis vinifera grapes contain AMAT-like acyltransferase (pAAT) transcripts. The arrow denotes positives: a 331 base pair product amplified with primers designed against the C-terminal region of the pAAT. Varieties in blue font were selected for cloning of a full length cDNA gene selected for cloning of a full-length cDNA gene.

His tag His tag T7 terminator XhoI (1) f1 origin putative AMAT-like gene sequence NcoI (663) NcoI (1350 ) k kan sequence enterokinase t ki S tag thrombin flPAAT in pet30(b)+ His tag 67 17 bp T7 promoter lac operator ColE1 pBR322 origin lac I