prof. alexander vainstein faculty of agricultural, …...vanillin o h o c h 3 eugenol o c h 3 trans...
TRANSCRIPT
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