Tal Isaacson, Fruit tree sciences, Newe Ya ar, ARO, ISRAEL RGC8

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1 Genetic and biochemical characterization of fruit from different apricot accessions highlights apricot as a rich source of phytoeneand phytofluene, and indicates CCD4 gene as a potential regulator of fruit color Tal Isaacson, Fruit tree sciences, Newe Ya ar, ARO, ISRAEL RGC8 June th, 2016

2 Carotenoids Essential components in photosynthesis, serve as precursors to phytohormones, furnish many fruit and flowers with bright colors and carotenoid degredation products are important aroma compounds. Essential in our diet as a source for vitamin A and considered as protective agents decreasing the risk of different diseases such as cancer and cardiovascular disorders. Important in determining fruit color, taste and nutritional value

3 OPP GGPP PPO + GGPP Psy Carotenoids in green tissues Pds 15-cis Phytoene 9,15-cis Phytofluene Pds 9,15,9 -cis ζ-carotene Z-Iso 9, 9 -cis ζ-carotene Zds 7, 9, 9 -cis Neurosporene 7, 9, 7, 9 -cis Lycopene (Prolycopene) Lcy-e δ-carotene Lcy-b α-carotene CrtR-b CrtR-e Zds CrtISO Lcy-b All-trans Lycopene γ-carotene Lcy-b β-carotene CrtR-b β-cryptoxanthin CrtR-b Lutein Zeaxanthin Zep O Antheraxanthin Zep O O Violaxanthin Nxs? Abscisic acid Xanthoxin Nced. O Neoxanthin

4 GGPP OPP + PPO Psy Pds 15-cis Phytoene GGPP Carotenoids in colored tissues 9,15-cis Phytofluene Pds 9,15,9 -cis ζ-carotene Z-Iso 9, 9 -cis ζ-carotene Zds 7, 9, 9 -cis Neurosporene Zds 7, 9, 7, 9 -cis Lycopene (Prolycopene) CrtISO Lcy-e All-trans Lycopene Lcy-b δ-carotene Lcy-b γ-carotene Lcy-b α-carotene CrtR-b CrtR-e β-carotene CrtR-b Lutein β-cryptoxanthin CrtR-b Zeaxanthin Zep O Antheraxanthin Zep O Abscisic acid Xanthoxin Nced O. Violaxanthin Neoxanthin Nxs? O

5 Apricot germplasm collection in Newe-Ya ar Apricot germplasmcollection includes about 85 accessions (imported and local ) and dozens of hybrid lines. The collection exhibit a wide range of phenotypes.

6 Carotenoids content and composition of fruit from the Newe-Ya ar apricot germplasm collection Fruit samples were collected from all accessions and analyzed by HPLC 100 Carotenoid content μg/g Fresh Weight phytoene phytofloune isomer ζ-carotene neurosporene lycopene isomer γ- carotene β-carotene β-cryptoxanthin esters unknown

7 Carotenoids content and composition of fruit from the Newe-Ya ar apricot germplasm collection Fruit samples were collected from all accessions and analyzed by HPLC 100 Carotenoid content μg/g Fresh Weight phytoene phytofloune isomer ζ-carotene neurosporene lycopene isomer γ- carotene β-carotene β-cryptoxanthin esters unknown

8 Carotnoids composition in apricot fruit is unusual Phytoene Phytofluene β-cryptoxanthin 1% Xanthophylls 5% unknown 3% ζ-carotene Neurosporene Lycopene phytoene 26% δ-carotene α-carotene Lutein γ-carotene β-carotene β-cryptoxanthin β-carotene 31% Zeaxanthin Antheraxanthin Violaxanthin Neoxanthin γ- carotene 2% lycopene isomer 5% ζ-carotene 2% phytofloune 25%

9 Carotnoids composition in Prunus fruit Phytoene Phytofluene ζ-carotene Neurosporene Apricot Lycopene δ-carotene α-carotene Lutein γ-carotene β-carotene β-cryptoxanthin Zeaxanthin Plum Antheraxanthin Violaxanthin Neoxanthin Peach Pictures: chefscottmcmillen.wordpress.com americanathleticinstitute.org

10 Choosing representing accessions for RNA-Seq Mustakawi Canino Paz µg/gfw β-cryptoxanthin lycopene isomer γ- carotene neurosporene ζ-carotene β-carotene phytofloune phytoene xanthophylls others

11 RNA-Seq RNA Extraction RNAseq Sample ID # Reads % of raw clusters per lane Mean Quality Score (PF) Mustakawi 1 33,363, Mustakawi 2 27,301, Mustakawi 3 34,043, Mustakawi 4 44,934, canino1 31,405, canino2 32,970, canino3 30,453, canino4 35,265, PAZ1 26,353, PAZ2 33,280, PAZ3 38,336, PAZ4 34,259, Undetermined 5,300,

12 Psy Phytoene Phytofluene ζ-carotene Neurosporene Lycopene δ-carotene α-carotene Higher expression of Psy-1 in Paz fruit Ggpp*2 Pds Pds Zds Zds γ-carotene β-carotene Apocarotenoids Psy Pds Lutein β-cryptoxanthin Zeaxanthin Zds Antheraxanthin 500 ABA Violaxanthin Neoxanthin Mustakawi Canino Paz

13 Higher expression of genes encoding carotenoid cleavage enzymes in Mustakawi Ggpp*2 Phytoene Phytofluene ζ-carotene Neurosporene δ-carotene α-carotene Lycopene γ-carotene β-carotene CCD NCED Mustakawi canino paz Lutein β-cryptoxanthin Zeaxanthin Apocarotenoids CCD4 Antheraxanthin ABA NCED Violaxanthin Neoxanthin Mustakawi canino paz

14 Genetic Association Studies for carotenoid traits in the Apricot Germplasm Collection % µg\gfw β Ph Phf Ly Tr β Ph Phf Ly Vis_c To_c Ccd Ccd CrtISO 1 CrtR-b Cyc Cyc Cyc Cyc Ipi 1 Pds Pds2 Psy Ptox SDG Zep Ziso

15 Summary HPLC analysis of fruit from more than 120 accessions: Great variation in carotenoid profile among accessions Apricot fruit exhibit unique carotenoidcomposition with high amounts of colorless carotenoidsand unusual isomers of lycopene. RNA-Seq of ripening fruit series: Elevated expression of genes encoding the first enzymatic steps in the biosynthesis pathway might contribute to high carotenoid content in the fruit. Elevated expression of genes encoding carotenoidcleavage enzymes might contribute to low carotenoidaccumulation in pale yellow fruit. Genetic association studies: Suggest CCD4as a potential regulator of Fruit carotenoidcontent.

16 Acknowledgment Matat Zohar Taly Trainin Keren Shemesh Einav Shor-Shimoni Adi Faigenboim Doron Holland Irit Bar-Yaakov Kamel Hatib Rotem Beja Zohar Ben-Shimhon Efraim Lewinsohn Einat Bar Ksenija Gasic (Clemson University) Amir Sherman Ada Rozen