THE 'GENECOASTER':

TOWARDS TAILOR-

MADE CROPS AND

COMPOUNDS

Prof. Alexander Prof. Alexander VainsteinVainsteinFaculty of Agricultural, Food and Faculty of Agricultural, Food and Environmental Quality Sciences,Environmental Quality Sciences,The Hebrew University of Jerusalem The Hebrew University of Jerusalem

INTIMACIES OF FLORAL SCENT

Plants secondary/specialized metabolites

• Products that are not essential for normal growth

and development of plants

• Natural products

• Multiple functions throughout the plant’s life cycle

Flowers fulfill their function…

Specialized metabolites dictate both the health and aesthetic value of plants

Importance of floral fragrance 5= most important, 4= very important, 3= important, 2= not very important, 1= not important at all

Prof. David H. Byrne, Department of Horticultural Sciences, Texas A&M

University. Online survey; 1439 responses; 2012

(+)-Carvone

O

CHO

OH

OCH

Vanillin

OH

OCH3

Eugenol

OCH3

trans-Anethole Benzaldehyde

CHO

Caraway

AlmondsAniseClovesVanilla

(-)-Carvone

O

Spearmint

The Rose Gardenothers

norisoprenes

aldehydes

acids

ethers

sesquiterpenes

other alcohols

oxygenated

monoterpenes(not alcohols)

aromatic ethers

monoterpenic

alcohols

aromatic alcohols

monoterpenehydrocarbons

hydrocarbons

Petal proteome during flower development

Constitutively expressed proteins ~ 6% of all unique spots

Stage-specific proteins ~ 30% of all unique spots

Timing of fluxes

Coordinated emission: temporal, spatial, diurnal

RT: 4.66 - 15.15 SM: 9B

5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

50000

100000

150000

200000

Re

la

tive

A

bu

nd

an

ce

0

10000

20000

30000

40000

50000

60000

70000

80000

Re

la

tive

A

bu

nd

an

ce

6.49

13.48

14.24

12.99

12.03 14.9210.50 11.099.477.78 9.077.37 8.605.55 6.235.05

12.83

6.60

13.50

9.4914.2512.0611.1010.509.797.14 9.058.557.646.134.93 5.45

NL:

8.51E4

TIC MS

wt1-

pYXVec

NL:

2.41E5

TIC MS

wt1-

pyxbamt2

Identification of genes

RT: 4.66 - 15.15 SM: 9B

5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

50000

100000

150000

200000

Re

la

tive

A

bu

nd

an

ce

0

10000

20000

30000

40000

50000

60000

70000

80000

Re

la

tive

A

bu

nd

an

ce

6.49

13.48

14.24

12.99

12.03 14.9210.50 11.099.477.78 9.077.37 8.605.55 6.235.05

12.83

6.60

13.50

9.4914.2512.0611.1010.509.797.14 9.058.557.646.134.93 5.45

NL:

8.51E4

TIC MS

wt1-

pYXVec

NL:

2.41E5

TIC MS

wt1-

pyxbamt2

VIRUSES YEAST

Maize DsRed

a

b

c

DFR DsRed235SP sgP nosT3’5’

sgP

g

e

d

f

0

0.5

1

1.5

N.alata WT DFR M2

OD

53

0/3

00

mg

FW

TGGCTTGTCATGAGACTCCTTG

5’ 3’

Tob acco Ratt le Vir usGe ne X

Ge

ne X Ge ne X

Ge ne X

VIGS – Virus Induced Gene Silencing

Tobacco Rattle VirusGene X

Maize DsRed

Silencing of Candidate Marker Genes for VIGS

CHS Silencing as a Marker for VIGS

CHS Target Gene

TRV2

BSMTCHS

ODORANT1CHS

Consensus Sequence of R2R3-MYB Domain

R2

R3

++ ++

*

*

*

* *

AR2

R3

++ ++

*

*

*

* *

R2

R3

++ ++

*

*

*

* *

A

Analysis of Floral Volatiles following VIGS of Candidate Genes

EOBs Are Developmentally and Spatially Regulated

EOBs Are Developmentally and Spatially Regulated

EOB (C) and IGS (D) promoter activity in petunia developing buds (a-c), flowers 2 dpa (d-f) and leaves (g-i) 36h after infiltration with Agobacteria carrying 35Spro:YFP and EOBIpro:DsRed or IGSpro:DsRed , respectively.

EO

BII

Rela

tive

exp

ress

ion

0

2

4

6

8

10

35S:E

OBII

35S:G

US

EO

BI

Rela

tive e

xp

ressio

n

0

5

10

15

20

A B* *

EOBII Overexpression in Petunia Flower

Buds Leads to EOBI Upregulation

EOBII Silencing

Results in EOBI

Downregulation

74 77 C

EO

BII

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

74 77 C

IGS

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

74 77 C

OD

O1

Rela

tive

exp

ress

ion

0.0

0.2

0.4

0.6

0.8

1.0

1.2

74 77 C

EO

BI

Rela

tive

exp

ress

ion

0.0

0.2

0.4

0.6

0.8

1.0

1.2

74 77 cEOBII-RNAi linecontrol

A

B

C

D

* *

*

*

**

**

JJ

B

benzy

l alc

ohol

phenyl

ethyl

alc

ohol

phenyl

ethyl

ace

tate

phenyl

ethyl

ben

zoat

e

eugen

ol

vanill

in

phenyl

acet

aldeh

yde

met

hylsa

licyl

ate

limonen

e

Em

iss

ion

g/f

low

er

x h

0.000

0.003

0.006

0.009

0.012

0.015

benza

ldeh

yde

met

hylben

zoat

e

isoeu

genol

benzy

lben

zoat

e

Em

issio

n

g

/flo

wer

x h

0.0

0.1

0.2

0.8

1.2C

36

41

44

B

***

***

***

***************

**** ******

Suppression of EOBI Expression

Reduces Levels of Volatile

Phenylpropanoids in Petunia Flowers36 41 44 C

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

EO

BI

Rela

tive e

xp

ressio

n

* **

EOBI-RNAi lines control

AE

mis

sio

n

g

/flo

we

r x

h

EOBI

EOBII

??

Suppression of EOBI Leads to Downregulation of the Expression of Genes in BOTH the Shikimate and Phenylpropanoid Pathways

36 41 44 C

DA

HP

SR

ela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* *

36 41 44 C

EP

SP

SR

ela

tiv

e e

xp

res

sio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* *

*

36 41 44 C

CS

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

**

36 41 44 C

CM

1R

ela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* **

36 41 44 C

PP

A-A

TR

ela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

** *

36 41 44 C

AD

T1

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

* **

DAHPS CS PPA-AT

EPSPS CM ADT1

36 41 44 C 36 41 44 C 36 41 44 C

trans-cinnamic acid

phenylacetaldehyde

C4H p-coumaric acid coniferyl alcohol

coniferyl acetate

CFAT

isoeugenol eugenol

vanillin

benzoic acidbenzaldehydebenzylacetatebenzyl alcohol

BPBT

benzoyl-CoA

benzylbenzoate

phenylethyl alcohol

phenylethyl acetate

phenylethylbenzoate

Phenylpropanoid PathwayShikimate Pathway

E4P

DAHPS

chorismate

EPSPS

CS

CM1prephenate

arogenate

ADT1

DAHP

PEP

shikimate

EPSP

PPA-AT

BSMTmethylbenzoate

L-phenylalanine

BPBT

36 41 44 C

PA

L1

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* **

36 41 44 C

BS

MT

1R

ela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

** *

36 41 44 C

EG

SR

ela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

36 41 44 C

PA

L2

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* **

36 41 44 C

BS

MT

2R

ela

tive

exp

ress

ion

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* *

36 41 44 C

PA

L2

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* **

36 41 44 C

IGS

Rela

tive e

xp

ressio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

**

36 41 44 C 36 41 44 C 36 41 44 C

PAL1 BSMT1 EGS

PAL2 BSMT2 IGS

IGS

Rela

tive e

xp

ressio

n

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Contr

ol

35S:E

OBI

BA*

a

b

c

d

EOBI Con

e

C ControlpD22-EOBI

PA

Lp

ro:G

LacZ

iIG

Sp

ro:G

LacZ

i

Control 35Spro:EOBI

D

Transcriptional activation of scent-related genes by EOBI

EOBI interacts with andactivates IGS and PALpromoters

35Spro:EOBI

PALpro:DsRed

35Spro:MYBYS

PALpro:DsRed

35Spro:CFP

PALpro:DsRed

35Spro:EOBI

IGSpro:DsRed

35Spro:MYBYS

IGSpro:DsRed

35Spro:CFP

IGSpro:DsRed

DsRed

CFP

PAL IGS

EOBI binds to and

activates its own

promoter

MBS1 MBS2

ODO1

EOBI

EOBII

Hierarchical Regulation of Floral Scent by MYBs

Scent-related gene expression

EOBIV

Emission versus Production

ph4

Glycosylation

Petal epidermis

35S:PAP1 Petunia x hybrida cv. ‘Blue-Spark’

Temperature effects on floral scent

Timing of fluxesdevelopmental signals

pig

men

t

00.0

0.2

0.4

0.6

0.8

01.0

Em

iss

ion

(g

/flo

we

r x

h) *

Emasculation Enhances Volatile Emission

GA levels in petunia corollas throughout flower development

Gibberellin Negatively Regulates Scent Production

Inte

rnal p

oo

l

g

/gF

W

0

5

10

15

20

25

30

35

07:001 dpa

17:00 07:002 dpa

17:00

*

*

*

*

benzald

ehyde

benzyl a

lcohol

phenyleth

yl alc

ohol

benzyl a

cetate

phenyl eth

yl aceta

te

eugenol

vanillin

isoeugenol

benzylb

enzoate

phenylaceta

ldehyde

hexylhexanol

limonene

pinene

Em

issio

n

g

/flo

wer

x h

00.00

00.01

00.02

00.03

00.04

00.05

00.06

00.07

00.08Control

GA3

*

*

*

*

*

*

*

*

*

Emission of Volatiles Volatiles in Internal Pools

anthesis 1 dpa

Em

iss

ion

g

/ fl

ow

er

x h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control GA10-7 GA10-6 GA10-5

Em

issio

n

arb

itra

ry u

nits

GA3 concentration [M]

10-7 10-6 10-5

GA3 concentration [M]

Control 10-6 10-5 10-4

00.0000

0.0005

0.0010

0.0015

0.0020

hexylhexanol hexylhexanol

GA3 concentration [M]

Control 10-6 10-5 10-4

00.0000

0.0005

0.0010

0.0015

0.0020

pinene pinene

GA3 concentration [M]

Control 10-6 10-5 10-4

00.000

0.001

0.002

0.003

0.004

0.005

limonene limonene

Gibberellin Affects Scent Production in a Dose Dependent Manner

Gibberellin Negatively Affects Scent Related Genes

BF

b

a

35S:GFP-R

GA

35S:GFP-P

hDELLA1 Flo

ure

se

nt

inte

ns

ity o

f G

FP

/RF

P p

er

ce

ll

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4(-GA)

(+GA)

-GA

+GA

RFP GFP

Flu

ore

sc

en

ce

in

ten

sit

y o

f G

FP

/RF

P p

er

ce

ll

DELLA proteins are involved in GA regulation of phenylpropanoidvolatile production

Food &

beverages

Bioreactors of

chemicals

Functional

analysis of

new TPSs

?

Crop

improvement

fe

c d

ba

fe

c d

ba

Shift of the Metabolic Flux

Malonyl-CoA

Leucoanthocyanidin

p-Coumaroyl-CoA

CHI

F3H

DFR

CHS

Flavanone

Dihydroflavonol

Chalcone

Anthocyanin

p-Coumaric acidCoA

C4H

4CL

Cinnamic acid

Phenylalanine

PAL

Time postanthesis (days)

Methylbenzoate Emission

Em

issi

on

(

g/f

low

er/2

4 h

r)

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

WT

Transgenic

Methylbenzoate

Benzoic acid

Metabolic Junctions

Malonyl-CoA

Leucoanthocyanidin

p-Coumaroyl-CoA

CHI

F3H

DFR

CHS

Flavanone

Dihydroflavonol

Chalcone

Anthocyanin

p-Coumaric acidCoA

C4H 4CL

Cinnamic acid

Phenylalanine

PAL

Benzoic acid

0

0.5

1

1.5

2

2.5

3

3.5

4

Control (pTRV2) 4CL 3' UTR supression

An

th

ocyan

in

s levels [O

D 530/100 m

g F

W]

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Benzaldehyde Benzyl alcohol Phenyl ethyl alcohol

Em

issio

n [

ug

/flo

wer*

24h

-1]

pTRV2 (Control)

4CL 3'UTR suppression

0

0.5

1

1.5

2

2.5

3

Control (pTRV2 Empty) 4CL 3'UTR Suppression

4C

L R

ela

tiv

e E

xp

ressio

n

Headspace of transgenic Petunia plants

expressing rose geraniol acetyl transferase

14 15 16 17 18 19 20 21 22

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Rela

tive

Ab

un

da

nc

e

2T

WT

13

4

5

1

2

3 4

1 benzyl alcohol 4 benzyl acetate

3 phenylethyl alcohol 5 phenylethyl acetate

2 methyl benzoate

0

50

100 41

53

69

80

93

107121 136

154

Time (min)

19.4 19.5 19.6 19.7 19.8 19.9 20.0 20.1

0

50

100

50

10019.68

19.76

19.76

19.72

Rela

tive

Ab

un

dan

ce

geranylacetate

T

WT

0

14.5 14.6 14.7 14.8 14.9 15.0 15.1

Time (min)

0

50

100

0

50

100

Rela

tive A

bu

nd

an

ce

14.99

14.46

14.46 14.99

T

WT

1-octanolacetate

40 210 3800

50

100 43

Volatiles produced by transgenic petunia and tobacco plants

(expressing rose geraniol acetyl transferase), following

feeding with various substrates

Carnation flowers with

linalool (Muscat wine)

GPP Linalool

LIS

LIS

14.0 14.5 15.0 15.5 16.0 16.5

1

2

3

Germacrene D

31

2

Leaves

Control

Leaves

T592-115

1

3

5

1

3

5

1 - tetradecanal

2 - silane (from fiber)

3 – hexadecane

Petunia with rose scent

8 10 12 14 16 18 20

0

50

50

Re

lativ

e A

bun

dance

50

100

+

+

+

+

8 10 12 14 16 18 20

0

50

50

Re

lativ

e A

bun

dance

50

100

+

+

+

+

Rela

tive a

bundance

100

Retention time (min)

Volatile compounds from PAP1 petunia

flowers

Benzyl alcohol

phenylacetaldehyde

benzaldehyde

p-cresol

methylbenzoate

Benzaldehyde 4 hydroxy

vanillin

Vanillin methyl ether

isoeugenol

:

0

1

2

3

4

5

6

Control Pap1-3 Pap1-2 Pap1-7

Em

iss

ion(

g*g

FW

-1*h

-1)

Em

iss

ion

(g*g

FW

-1*h

-1)

Control Pap

1-3

Pap

1-2

Pap

1-7

0

1

2

3

4

5

6

Control Pap1-3 Pap1-2 Pap1-7

Em

iss

ion(

g*g

FW

-1*h

-1)

Em

iss

ion

(g*g

FW

-1*h

-1)

Control Pap

1-3

Pap

1-2

Pap

1-7

Enhanced pigmentation and levels of floral volatiles

in Pap1 transgenic rose

Orcinol O-methyltransferase expression

in Rose petals

0

50

100

150

200

250

300

350

400

450

500

control pap 11 pap 12

Re

lati

ve

ex

pre

ss

ion

Phenylacetaldehyde synthase expression

in Rose petals

0

100

200

300

400

500

600

control pap 11 pap 12

Re

lati

ve

ex

pre

ss

ion

Orcinol O-methyltransferase expression

in Rose petals

0

50

100

150

200

250

300

350

400

450

500

control pap 11 pap 12

Re

lati

ve

ex

pre

ss

ion

Phenylacetaldehyde synthase expression

in Rose petals

0

100

200

300

400

500

600

control pap 11 pap 12

Re

lati

ve

ex

pre

ss

ion

Aromatic yeast

TPS

HMG1/2

Overexpression of

FPS from A. thaliana

Knockout of

DPP1 & LPP1

pyrophosphatases

Addition of

truncated

trHMG-R into

chromosomeTrans. factors

upc2-1 (mutated)

SUT1

MET3p-ERG9

TFS

Fusion

enzyme

XFarnesol

Expression of

GPS from A.

thaliana

X

sesquiterpenessesquiterpenes

monoterpenesmonoterpenes

TPS

HMG1/2

TPS

HMG1/2HMG1/2

Overexpression of

FPS from A. thaliana

Overexpression of

FPS from A. thaliana

Knockout of

DPP1 & LPP1

pyrophosphatases

Addition of

truncated

trHMG-R into

chromosomeTrans. factors

upc2-1 (mutated)

SUT1

MET3p-ERG9

TFS

Fusion

enzyme

XFarnesol

XFarnesolFarnesolFarnesol

Expression of

GPS from A.

thaliana

X

sesquiterpenessesquiterpenes

monoterpenesmonoterpenes

Strain (BDXe)

iCstps

1

imtC

stps

1

tHM

G+i

mtC

stps

1

tHM

G+i

Cstps

1+im

tCstps

1

tHM

G+m

tFPS

+iCstps

1+im

tCstps

1

Va

len

cen

e (a

rbit

rary

un

its)

0

1e+6

2e+6

3e+6

4e+6

TPS

HMG1/2

Overexpression of

FPS from A. thaliana

Knockout of

DPP1 & LPP1

pyrophosphatases

Addition of

truncated

trHMG-R into

chromosomeTrans. factors

upc2-1 (mutated)

SUT1

MET3p-ERG9

TFS

Fusion

enzyme

XFarnesol

Aromatic yeast

Expression of

GPS from A.

thaliana

X

sesquiterpenessesquiterpenes

monoterpenesmonoterpenes

TPS

HMG1/2

TPS

HMG1/2HMG1/2

Overexpression of

FPS from A. thaliana

Overexpression of

FPS from A. thaliana

Knockout of

DPP1 & LPP1

pyrophosphatases

Addition of

truncated

trHMG-R into

chromosomeTrans. factors

upc2-1 (mutated)

SUT1

MET3p-ERG9

TFS

Fusion

enzyme

XFarnesol

XFarnesolFarnesolFarnesol

Aromatic yeast

Expression of

GPS from A.

thaliana

X

sesquiterpenessesquiterpenes

monoterpenesmonoterpenes

~130 mg/l

plant terpene

TPS

control

endogenous sesquiterpene endogenous sesquiterpene

control

12.8 13.0 13.2 13.4 13.6 13.8 14.0 14.2 14.4 Time (min)

0 20

40

60

80

100 0

20

40

60

80

100

13.91

13.27

13.97 13.51 13.59 14.03 14.38 14.25 13.11 13.00 12.85

13.27

13.35 13.58 13.76 14.24 13.99 13.20 14.45 12.77 13.08

NL: 1.79E5 m/z= 160.5-161.5+ 188.5-189.5 F: MS 200207-spme-ads

NL: 3.09E5 m/z= 160.5-161.5+ 188.5-189.5 F: MS 200207-spme- vec-pmy5

Plant sesquiterpene

Aromatic yeast

TPS

HMG1/2

Overexpression of

FPS from A. thaliana

Knockout of

DPP1 & LPP1

pyrophosphatases

Addition of

truncated

trHMG-R into

chromosomeTrans. factors

upc2-1 (mutated)

SUT1

MET3p-ERG9

TFS

Fusion

enzyme

XFarnesol

Expression of

GPS from A.

thaliana

X

sesquiterpenessesquiterpenes

monoterpenesmonoterpenes

TPS

HMG1/2

TPS

HMG1/2HMG1/2

Overexpression of

FPS from A. thaliana

Overexpression of

FPS from A. thaliana

Knockout of

DPP1 & LPP1

pyrophosphatases

Addition of

truncated

trHMG-R into

chromosomeTrans. factors

upc2-1 (mutated)

SUT1

MET3p-ERG9

TFS

Fusion

enzyme

XFarnesol

XFarnesolFarnesolFarnesol

Expression of

GPS from A.

thaliana

X

sesquiterpenessesquiterpenes

monoterpenesmonoterpenes

Jasmine Slilat, Oded Skaliter, Gony Dvir, Noam Schpaer, Yuling Yue, Orit Edelbaum, Elena Shklarman, Marianna Ovadis, Elena Markevka, Tania Masci, Alon Cnaani, JavieraAravena Calvo, Ben Spitzer, Moran Farhi, Michal Ben Zvi Moyal, Nadav Ketraouro, EnoshTomer, Or Shalev; Ira Marton, Arik Honig, Amir Zuker, Aelet Omid, Barry Cohen, Michal Rosenthal Thank you