The Effects of Ultraviolet Radiation and Canopy Shading on Grape Berry Biochemistry & Molecular Biology Professor Brian Jordan Professor of Plant Biotechnology Agriculture and Life Sciences Faculty Lincoln University

Responses of Plants to Light Light PhotosynthesisSugars other organic compounds Information leaf growth stem growth germination, etc. flowering dormancy plant habit, etc. direction of growth Small amounts of light Daily duration of light Direction of light

Plants Red & far red Blue UV-AUV-B

Ultraviolet Penetration through the Stratospheric Ozone Layer UV-A nm UV-B nm UV-C <280nm O 3 layer 0% 100% Earth’s surface PAR 700nm – 380nm

Photoperception to gene expression Photoperception Signal Transduction Gene Expression

UV-B Photoreceptor UV-B Specific Photoreceptor Signal Transduction Non-Specific Via ROS Via DNA damage Changes to gene expression

H2O2H2O2 PR genes JA O2-O2- PDF1.2 Ethylene SA Transcription factors Photosynthetic genes H2O2H2O2 Chloroplast signal, electron transport/ photophosphorylation UV-B Peroxidase NADPH oxidase Receptor Signal Transduction Pathways ? NO Ca 2+ /CaM Phosphorylation NOS Chs

Role of UV/Light in Grape Development and Wine Quality Effect on “ageing” of white wines in New Zealand Changes to polyphenolic compounds Changes to amino acids/protein content Impact on aroma/flavour (methoxypyrazines) Lipoxygenase as an example of molecular approach

Vineyard experiments UVA+, UVB+ screen UVA+, UVB- screen UV- screen No frame No leaf removal, no frame UV+ UVA+ UV-

UV-B DamageNo UV-B Damage

UV-absorbing compounds

Amino Acid Metabolism and Implications for Wine Industry UV (and PAR) NITROGEN (Uptake and assimilation) AMINO ACIDS Methoxypyrazines: amino acids as precursors to flavour and aroma compounds Phenolics: amino acids as precursors – implicated in ageing and bitterness in white wine Amino acid composition and implications for fermentation bouquet and yeast assimilable nitrogen Glutathione: implicated in the prevention of browning process Valine, isoleucine, leucine Phenylalanine, tyrosine, tryptophan All amino acids except proline Cysteine, glutamate, glycine

Amino Acid Composition Glutamine Proline Arginine Alanine Serine Glutamate Arginine Proline Glutamine Alanine Threonine Serine Increasing Amounts Chardonnay Sauvignon blanc

Light regulation of nitrogen metabolism Light regulates the conversion of glutamate into glutamine in the chloroplast This involves the GOGAT pathway and requires ATP This assimilation of nitrogen then provides amino acids/amines to the fruit Glutamate Glutamine

Amino acids

Major aroma chemicals 3-mercaptohexanol/3- mercaptohexanal acetate –Tropical fruit and Citrus aromas Methoxypyrazines –Green/green-pepper or capsicum aromas

Present Understanding: Synthesis of Thiol Precursors Lipids and Fatty Acids in Cell Membranes 5/6 Carbon Backbone eg, s-3- (hexan-1-ol)- Glutathione LOX HPL etc Non Volatile s-cysteine Conjugate Precursor Grape Metabolism through Berry Development and in Response to the Environment Changes during Must Fermentation Release of Aroma Volatiles Primarily by Yeast VERAISON Hard Solid Berry Soft Berry at Harvest ‘Membrane Turnover’ GSTs

LOX-HPL pathway

13-LOXs Type I 9-LOXs Type I Type II13- LOXs L O X 1 G m 1 L O X 1 G m 2 L O X 1 A h 1 L O X 1 P s 2 L O X 1 G m 6 L O X 1 G m 7 L O X 1 G m 3 L O X 1 P s 3 L O X 1 L c 1 L O X 1 G m 4 L O X 1 G m 5 L O X 1 C s 1 L O X 1 C s 2 L O X 1 S t 2 L O X L V v L O X 1 A t 2 L O X 1 S t 1 L O X 1 L e 1 L O X 1 N t 1 L O X 1 P r d 1 L O X 1 A t 1 L O X 1 C a 1 L O X M V v L O X B V v L O X C V v L O X 1 H v 1 L O X 1 Z m 3 L O X 1 O s 1 L O X 1 Z m 1 L O X 2 Z m 6 L O X D V v L O X 2 A t 2 L O X 2 A t 3 L O X 2 S t 2 L O X O V v L O X R V v L O X 2 A t 4 L O X P V v L O X 2 O s 1 L O X 2 Z m 1 L O X 2 H v 1 L O X 2 O s 2 L O X 2 A t 1 L O X 2 B n 2 L O X 2 S t 1 L O X 2 P o d 1 L O X 2 P o d 2 L O X J V v L O X K V v L O X A V v L O X E V v L O X F V v L O X G V v L O X H V v L O X I V v Phylogenetic analysis of grape LOXs and characterised LOXs from other plants

Proportional distribution of grape LOXs in different berry fractions Relative expression of four berry expressed LOXs SB berry expressed LOXs

Relative gene expressions of berry expressed LOXs during development

Relative gene expressions of berry expressed LOXs during upon wounding

I – berries with obvious signs of infection, NI – berries closely located to the infected, Control – healthy berries distantly located from the infected. Relative LOX gene expressions in SB berries infected with Botrytis

pH effect on recombinant VvLOXA activity

pH effect on recombinant VvLOXO activity

Methoxypyrazines Little is known about their biosynthesis –Thought to derive from amino acid biosynthesis Accumulate up until veraison Degrade after veraison and with exposure of grape bunches to light At low concentrations (ng.L -1 ) contribute to green/green- pepper aromas

UV responses & wine quality

+UV No leaf No No No UV removal frame UV-B UV responses & wine quality

Effects of UV and Leaf Removal on Wine Quality Methoxypyrazine levels low in juice at harvest, but high early in grape development: control of gene expression from amino acid precursors Amino acid composition different in juice in response to light environment Regulation of proline biosynthesis important for fermentation Flavonoids accumulate with UV exposure: role of transcription factors Lipoxygenase pathway: complex gene family and expression pattern

Acknowledgements Grape Biotechnology and UV Research Jason Wargent, Lancaster University, UK Scott Gregan Stephen Stilwell Andriy Podolyan (Ph.D.) Jim Shinkle, Trinity University, USA Dr Rainer Hofmann Dr Chris Winefield Professor Brian Jordan (Programme Leader) Support From: Foundation for Research, Science & Technology NZ Royal Society/MoRST COST-ACTION 858 Marlborough Wine Research Centre, Auckland University & Plant & Food Research New Zealand Wine Industry Lincoln University