Genetic improvement of

tomato fruit quality attributes

G. Diretto, March 8th

Class Isomers (n) Unique (n)

Polar 1,600 1,000

Non Polar 400 200

Untargeted analysis

Manual curation

and database interrogation

Differentially

accumulated

Ions (accurate

mass)

List of putative identified

compounds

Mass chromatogram

Validation (literature, auth. Std.,

Isotopic pattern, MS/MS)

Relative amounts (Fold Internal Std)

Targeted analysis (known primary and secondary

metabolites)

LC-MS

(Orbitrap) GC-MS

(Triple Quadrupole)

Targeted analysis of

semi-volatiles and

volatiles

Metabolomics Pipeline at

Tomato

Fruit

Grape Berry

Chlamydomonas

reinhardtiii

Rabbit

Tomato

Fruit

Potato

Tuber

Wheat

grain

Maize

kernel

Grape Berry

Saffron

Stigma

Eggplant

Fruit

Garlic

Clove

Nannochloropsis

spp.

Hazelnut Seed

Leaf

(Arabidopsis, Tomato,

Potato, Wheat)

Pepper

Fruit

Orchid

spp.

Orange Fruit Strawberry

fruit

Olive Fruit

Tomato fruit: a well-known source of pro-nutritional molecules

Carotenoids (lycopene)

Vitamin C

Low calories Low fat (0 cholesterol)

Potassium

VITAMINS:

- Thiamin;

- Niacin;

- Riboflavin.

MINERALS

- Iron;

- Calcium;

- Manganese.

Average levels in: Fibers

Vitamin K

Folates

Flavonoids

but..."All that glisters is not gold" (by W. Shakespeare)

Glycoalkaloids Allergenes

Increasing or Pyrimiding multiple quality fruit attributes

by OX and GE strategies

NEGATIVE

- GLYCOALKALOIDS;

- - ALLERGENS.

POSITIVE

- CAROTENOIDS;

- STEROLS, SAPONINS.

The metabolic engineering of carotenoids in tomato at ENEA

lycopene

-carotene

α-carotene

LCY-e

LCY-b

BETA

β-carotene

LCY-b

BETA

Wild-type

HO

OH

lutein

CYP97A

CYP97C

Pds LCY-b Nos

Rosati et al., 00

Pds CHY Nos

Pds LCY-b Nos

+

Dharmapuri et al., 02

Pds CHY Nos

Diretto et al.,

Unpublished OH

HO O

O

??? OH

HO OH

O

neoxanthin

violaxanthin

CHY, CYP97A

ZEP

HO OH COOH

ABA

Transcriptomics Metabolomics Phenomics

Carotenoids - antioxidants

- pro-vitamin A

Alteration at sub-cellular and ”local” metabolism level

Pds LCY-b Nos

Rosati et al., 00

GGPP

Sterols

Tocochromanols

Quinones

GPP

down-accumulation up-accumulation

Lyc-apos

b-car-apos

Lycopene

Phytoene/Phytofluene

a-/b-d-/carotene

b-b-xanthophylls

WT LCY-b

EM of LCY cromoplast

Several primary and secondary (mainly phenylpropanoid) metabolic

classes are altered in LCY ripe fruits

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

FO

LD

WT

WT

LCYb1

LCYb3

Amino acids

Phenylpropanoids

Extended shelf-life of “Golden” tomatoes

Wt LCY-OX

Cell Wall

Methyl-esterified pectins

(Immunodot)

Cell wall and cuticle are strongly altered in “Golden” fruits

Total Cell Walls

Galacturonate

LCY1 Wt

0

1

2

3

4

5

6

mg

/cm

2

WT LCY1

Cuticle

Waxes

“Golden” fruits show reduced ethylene production

MG B B+4 B+10 B+15

0

2

4

6

8

10

Ethylene production

mL x

h-1

x g

m(F

W)-

1

WT LCY-b1 LCY-b2 LCY-b3

Stage of ripening

ABA Ethylene Genotype

hp-3 mutant (Galpaz et al, ‘08)

nced1 RNAi (Sun et al, ‘12)

LCY-OX (this work) ?

Could ABA be the missing link between carotenoid content and ethylene/ripening?

Cross-link between Ethylene and ABA

ABA increases up to >10-fold in “Golden” fruits and

is responsible of the extended shelf-life

ABA production

mg /

gm

DW

0

100

200

300

400

MG B B+4 B+10 B+15

WT

LCYb1

LCYb2

LCYb3

0

20

40

60

80

100

120

WT

LCYb1-

mock

LCYb1-

abam

ine

LCYb3-

mock

LCYb3-

abam

ine

Sh

ore

Abamine treatment

violaxanthin

neoxanthin ABA

**

**

Fruit Firmness

Correlation network analysis

of the main regulatory “hubs” in “Golden” fruits

Negative Correlation

Positive Correlation

Up-regulation

Dw-regulation ns=node strength= AVG

n=number of nodes

• Node size according ns • Only correlations >0.90 are shown

Gene

Metabolite

Phenotype

Enzyme

• Edge width according

NS=network strength= AVG ns

Network strength=097

Nodes=1566

||>0.90

Ethylene

Lycopene

β-carotene ABA

Ethylene

Lycopene

NAC-NOR

CNR

β-carotene

HB-1

ABA

RIN

AP2a

TAGL1

TDR4

AP2a RIN NAC-NOR

ABA

N=1008

NS=0.97

0.90 0.95 0.99 ABA

ACOL

CUL4

NAC-NOR

MAPK20-1

C3H23

TDR4

CBL

CNR

ER

SAPL

stig

14-3-3

CIPK17

zea

MAPK16

SENP1B

LEA3

RAB11A

EIN2

SDR

DNAJ20

nar-C3H7O2NS

ACS6

C3H22

NR

LEA4

IDI TCP

HSP20

DFR

CBF2

bcar

ethy

TLP

cutthick

XYL2

ACS4

EXP1

SENP2

APC1

14-3-3

14-3-3

neo

DASS

IP3P

IBR

api6,8digal

CWM

PL

AQP

CCD1A

BTF3L

nar-hex

nar

AQP1

TAGL1

EF-CAML

EF-CAM

CAMBP

H2A

K-RHA-

GAL-GLU

pha

7OH-ABA

NCED RIN viola

HB-1

SIXTH8

lyc

DGAT

ARF1

AP2A ERF13

ISPF

FPS

GGPS2

FLUL

CBL EF-CAM

EF-CAM

CAMBP

EF-CAML CAMTA2

CIPK16

C2

C2

C2MTP

SLT1

ATPS

SULTR2

SULT

SUFE

ITPK1

AQP

AQPL

A-DOX

A-DOX2

MAPK3

MAPK18

MAPK

ROP-GEF

MBF1

ZRANB2

ABI3 BZIP1

C3H53

C3H6

WRKY78

EPFL4

KINL

KINL

CA

HSP40

DNAJ16

DNAJ10

OLP OLP

ASR

ERF2b

ERF3

ERF2B

ERF9

ERF1

ERF4

ARF

ARF9

ARF16

SAUR

AE

CLPP

PP2C

PP2C

PP2C

PP2C

PX

ADC PL

pro

GST KDBP

HMA

SMT

PRP

STK

SEUSS

GPI1

AE

SAMDC

PPR

GSK

LHLP

ETO1 gal DGAT

CSL KDPB

OPT

VDAC

SLP

CBS

PRMT

BZIP1

CNOT7

NIA

FIP1

NDPK

QR

ACO1

DOB1

TIR-NBS

SP

LOX

phe HP-1

PEPT1

HPT

SRC2

PPR

SLC

MRC

PHI-1

PR-4B MSF fer

SDR3

PPR

GRX

PIP

PSMC6B

PIK

HMA

HP-1

CASP

SUMO

NAC

CTSBL

TRXH2 KCS

PPR

PAP

ENO

ASR4

AXE-A

DI19-3

AGO4

GSTL

ZEP

ILKAP

FIP1

ANK

PDK

G6PDH

meli

GPI

HSP17.6

SAMS

PPR

ACS

GRP

XYL1

BG

DGK

AP

FABP

PRMT

CHLM

SEC14L

MDTK

TCTP

DRP

BI

GAPDH

NAP1L-4

MCM4

put

AGO4 TAG1

PPR

GRX

BZIP1

MDH

PLP3

UBC11

CHD

LCY-B

HCT

DFR

PPASE

ABH1

RH5

NIA

HLMT

BG

H2BL

ST225

AOC

PHI-1

suc

ADP NF-YA3

WD40-18

CC-NBS-LRR

FRKL1

CYP

HPA HRGP

PHD

nar7glu

NAC

PAT PSMC6B

PAO

SYN

AP2/B3 MAS

coum

chlor

LEA3

LOB1

PGM

PSMC6B

KARI

HSP20

EXP

PSMC6B

GST

EF-CBP

SRP ARR10

BCAT NMNAT1 HDAC2L

CYN

fru6P

ACS2 ACO

HEX

wl

PEPC

LSD1l

NRP

DCP1L

NAC

TTC38 fleshfirm

DFR

UGCG

PPR

PPR

NBS-LRR

F3H

MDTK MBP

CPK

Conclusions - Carotenoids

Overexpression of lycopene b-cyclase results in the following phenotypes:

Increased β-carotene, b-b-xanthophyll content, and deep isoprenoid remodeling;

Large primary and secondary metabolome remodelling;

Increased shelf life, with several components likely involved (increased cell wall and

cuticle; reduced ethylene);

Increased ABA levels, which is mainly responsible of this phenotype (reverted when

fruits are treated with abamine);

The mechanism looks to rely on a complex cross-link between ABA and ethylene.

Antinutritional targets

Glycoalkaloids (GA)

Allergenes (AL)

- gastrointestinal and neurological disorders;

- disruption of membranes;

- inhibition of acetylcholine esterase activity.

- food allergy increased during

the last decades;

- affects 3-4% of the adult population

and 5% of children;

- 1.5% in Northern Europe up to 16%

in Italy.

Itkin et al., ’13

Genome editing (GE) for GA and AL

Vazquez-Vilar et al., '16

+1 T Insertion

WT

Cut site

To

T1

T1 PAM

PAM

PAM

PAM

Biallelic

-8 Deletion

+1 Insertion

CAAGATAC

% GE % Biallelic % Homozygous % Heterozygous

GA1 85.7 66.7 20.0 13.3

AL1 82.9 60.3 22.4 17.2

GE efficiency in GA/AL lines

0

2000

4000

6000

8000

10000

12000

L1L10

L12A

L13A

L13BL19

L4B

L5B

L6A

L6B L7

L9L9A

L9B

WT

FoldIS

Tomatidine-4-hexose

Tomatidine-2-hexose-2-pentose

Tomatidine

Tomatidenol

Hydroxytomatine

EsculeosideB

EsculeosideA

DehydroesculeosideA

δ-Tomatine

γ-Tomatine

β-Tomatine

Acetoxy-tomatine

α-tomatine

Glycoalkaloid contents in edited lines

0.00

0.01

0.02

0.03

0.04

0.05

L1 L4B L6A L9B L13A WT

FoldIS

Tomatidine+4xhexose

tomatidine+2hexose+2pentose

Tomatidine

Tomatidenol

LycoperosideG-OH

hydroxytomatine+

Dehydroesculeoside_A_FA

Acetoxyhydroxy-Dehydro-tomatine

Acetoxy-tomatine

δ-Tomatine

γ-Tomatine

β-Tomatine

a-tomatine99.23 – 99.99%

73.02 – 99.98%

Additional bonus features in edited fruits

- cholesterol lowering;

- cancer prevention;

- skin protection. 0.00

0.05

0.10

0.15

0.20

0.25

0.30

L1 L10L12AL13AL13BL19L4BL5BL6AL6B L7 L9 L9AL9BWT

FoldIS

Stigmasterol

- reduction in atherogenesis;

- metabolization into pregnanes (putative

anti-osteoporosis, anti-menopausal disorder,

and antitumor actions).

0.00

1.00

2.00

3.00

4.00

5.00

6.00

L1 L10L12AL13AL13BL19 L4B L5B L6A L6B L7 L9 L9A L9B WT

FOLD

IS

Uttroside B

Itkin et al., ’13

Conclusions – GA and AL

Efficient GE was achieved in multiple GA and AL targets;

GA reduced up to 99.98% in edited leaves and fruits;

Edited fruits also yield higher levels in pro-nutritional compounds, as saponins and

phytosterols.

Acknowledgements

A. Fernie

S. Frusciante

A. Fiore

G. Giuliano

J. Giovannoni

J. Rose

B. Mattei R. Jetter

Carotenoids Gycoalkaloids/All

ergenes

A. Fiore

F. Sevi

C. Lico

A. Granell

D. Orzaez

Thank you for the attention!

contact:

gianfranco.diretto@enea.it