creating new tomato varieties with high-value …...expansin pc-esterase psy1 acyl-coa synthetase 5...
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Creating new tomato varieties with high-value
carotenoids through transgenics and non-GMO
approaches
Joseph (Yossi) Hirschberg
Department of Genetics, Alexander Silberman Institute of Life
Sciences, The Hebrew University of Jerusalem
7-8.3.2019
Cathie Martin (2018) A role for plant science in underpinning the
objective of global nutritional security? Ann.Bot. 122:541-553.
Processes impacting plants and plant-based foods in the diet
Theorized mechanisms of action of carotenoids and chronic diseases in which carotenoids may play a role.
Cooperstone and Schwartz (2016) Handbook on Natural Pigments in Food and Beverages. http://dx.doi.org/10.1016/B978-0-08-100371-8.00020-8
HO
OH
OH
HO
HO
OH
O
O
HO
OH
OH
O
O
OH
O
OH
Lycopene
Zeaxanthin
Lutein
-Carotene
Violaxanthin
Neoxanthin.
Capsorubin
Common carotenoids
GDP
FDP
GGDP
Cytokinins
Monoterpenes
Triterpenes
Chlorophylls
Tocopherols Phytol
Carotenoids
Sesquiterpenes
Sterols
Farnesylated proteins
Biosynthesis of carotenoids
(C5)
(C10)
(C15)
(C20)
(C40)
Abscisic acid (ABA)
Strigolactones
MEP pathway
PSY
DXS
IDP + DMADP
Diterpenes, Ubiquinone
Gibberelins, Phylloquinone
Plastoquinone, Tocotrienols Other signaling
Apocarotenoids
Deciphering the
carotenoid pathway
Through mutations
Map-based cloning of
pigmentation mutations
z (2)
r (3)
t (6)
B, og DEL
wf (2)
lut
hp3 (2)
NFZR
CPTAR
fcd1(4)
nxd1 (2)
Navot Galpaz, Naama Menda, Dani Zamir
The carotenoid
pathway in plants
(2019)
NSY
PDS
CRTISO
phytoene
lycopene
prolycopene
ZDS
tri-cis-ζ-carotene
di-cis-ζ-carotene
ZISO
β-carotene
β-xanthophylls
GGDP IDP+DMADP
LCYB
HYDs
LCYB, LCYE
α-carotene
HYDs
lutein
PSY2 MEP pathway
80
60
20
0
40
Xanthophylls -carotene
Phytoene
Lycopene
MG B O P R IM
Carotenoid composition in fruits
Ca
rote
no
id c
on
ce
ntr
ation
[μ
g.g
−1 F
W]
Stages of fruit development
Regulation of carotenoid biosynthesis in fruits
(Tomato)
DXS
PSY1
PSY2
CYCB X X
PDS
CRTISO
phytoene
lycopene
prolycopene
ZDS
tri-cis-ζ-carotene
di-cis-ζ-carotene
ZISO
β-carotene
β-xanthophylls
GGDP IDP+DMADP
LCYB
HYDs
LCYB, LCYE
α-carotene
HYDs
lutein
PSY2 MEP pathway
Regulation of carotenoid biosynthesis in fruits
(Tomato)
DXS
PSY1
PSY2
CYCB X X
Liu et al. Plant Physiol. 2015
Regulation of fruit ripening in tomato
PDS
CRTISO
GGDP
Phytoene
Lycopene
Prolycopene
ZDS
tri-cis-ζ-Carotene
di-cis-ζ-Carotene
Neurosporene
ZDS
ZISO
tangerine t =
The phenotype of the double mutant
yellow-flesh (r) / tangerine (t) is that of
tangerine (accumulation of tetra-cis
lycopene, ‘prolycopene’).
Jenkins and Mackinney, Genetics 1953; Tomes et al. Nelson, and North, Genetics 1953
Epistasis of t over r
β-Carotene
tangerine is epistatic over yellow-flesh
PSY1 = yellow-flesh r2997
PDS
CRTISO
GGDP
Phytoene
Lycopene
Prolycopene
ZDS
tri-cis-ζ-Carotene
di-cis-ζ-Carotene
Neurosporene
ZDS
ZISO
tangerine
zeta
=
=
=
1. The mutation in r2997 abolishes
transcripts of the Psy1 gene.
2. Loss-of-function mutation in CrtISO
restores transcripts of Psy1 in r2997.
3. The signal which restores expression
of Psy1 is derived from neurosporene
and/or prolycopene, probably a cis-
apocarotenoid.
Epistasis of t over r
CCD?
β-Carotene Kachanovsky*, Filler* et al., PNAS, 2012
yellow-flesh r2997 PSY1
The molecular basis of t over r epistasis
Cis-carotenes derived signal affect gene expression (Kachanovsky, Filler et al. 2012, PNAS)
An apocarotenoid-derived signal regulates leaf
development (Avendano-Vazquez et al. 2014, Plant Cell)
An uncharacterized carotenoid derivative regulates
periodic root branching (Van Norman et al. 2014, PNAS)
The apocarotenoid metabolite zaxinone regulates
growth in rice (Wang et al. 2019, Nature Comm.)
Apocarotenes affect gene expression
What is the molecular basis of yellow-flesh r2997?
Kachanovsky*, Filler* et al., PNAS, 2012
Italian physician and botanist Pietro
Andrea Mattioli (Matthiolus) in his
1544 Commentaries refers to the tomato
as a “Poma aurea” (golden apple),
which is generally taken as evidence
that yellow tomatoes preceded their red
counterparts across the Atlantic.
"Pomi d'oro“/ golden apples/ Pomodoro
The origin of the yellow tomato (yellow-flesh)
Pietro Mattioli
The tomato page from the 1590 edition
H. L. Price, A. W. Drinkard and Jr. (1909) The Plant World, 12:10-18
The mutation yellow-flesh (r)
Expansin PC-Esterase PSY1 Acyl-CoA synthetase
1 2 3 4 5 6 7 8 9
III IV V VI VII VIII
1 kb
75% 25%
ATG RIN
Chromosome 3
5 kb
Structure of the gene Psy1
Varda Mann
The genetic basis of yellow-flesh r2997
1 2 3 4 5 6 7 8 9
III IV V VI VII VIII
4.8 kb retrotransposon Rider/Kielia
Expansin PC-Esterase PSY1 Acyl-CoA synthetase
5 kb
Uri Karniel
1 2 3 5
1 3 5
1 5
40% 18% 15%
15% 18% 35%
35% 64% 10%
25%
15%
1 2 3 5 I
1 2 3 5 I II
r2997 r2997 r2997 /t3406 /t3002
1 2 3 4 5 6 7 8 9
III IV V VI VII VIII
4.8 kb retrotransposon Rider/Kielia
The genetic basis of yellow-flesh r2997
Bi-color phenotypes
Itay Zemach, Shdema Filer
The genetic basis of Bicolor
Expansin PC-Esterase PSY1 Acyl-CoA synthetase
5 kb
1 2 3 4 5 6 7 8 9
III IV V VI VII VIII
1 kb
Deletion (3.8 kb)
90%
ATG
• Fruit with yellow-red segments across the entire fruit.
• Genetically inherited.
• No sign for this segmentation on green stages of fruit development.
Arlecchino, another allele of yellow-flesh
Itay Zemach and Orly Dery
1 2 3 4 5 6 7 8 9
III IV V VI VII VIII
3.9 kb hAt transposone
Expansin PC-Esterase PSY1 Acyl-CoA synthetase
5 kb
Uri Karniel
An hAT-type transposon in Psy1 (rArlecchino)
Transposase gene
Early and late somatic mutations may occur in the same fruit
What activates the Arlecchino transposition?
Three main strategies to manipulate carotenoids in
tomato fruit:
• Increase the biosynthetic flux
• Increase the storage capacity (accumulation)
• Metabolic engineering for novel carotenoids
1 China 52.6
2 India 18.7
3 United States 14.5
4 Turkey 11.9
5 Egypt 8.3
6 Iran 6.0
7 Italy 5.6
8 Spain 4.9
[million tons]
Total world 170.8
Tomato production in 2017
http://www.tomatonews.com/en/background_47.html
Global processing tomato production in 2017
high-pigment 1 (hp1) in a tomato UV-DAMAGED DNA-BINDING
PROTEIN 1 (DDB1) homolog
high-pigment 2 (hp2) in DETIOLATED 1 (DET1) homolog
Levin et al. Isr J Plant Sci, 2006
WT
hp2
Lycopene concentration
in hp2 is 70-80 percent
higher than in WT
Increase the storage capacity (accumulation)
Haematococcus pluvialis
OH
OH
O
OOH
OH
O
OZeaxanthin
Astaxanthin
Canthaxanthin
-Carotene CrtOCrtR-b
CrtR-bCrtO
Metabolic engineering of astaxanthin biosynthesis
Silencing Lycopene
LCY-B
d-Carotene
LCY-B
Zeaxanthin
Lutein
Antheraxanthin
z-Carotene
Phytoene
GGPP
ZDS
PDS
PSY1
-Carotene
CRTR-B2
DMAPP
VDE
IPP IPI
Violaxanthin
GGPS
(3xIPP)
Neoxanthin
NXS
CRTR-B
CRTR-E
LCY-E
CYC-B
ZEP
VDE
PSY2
CRTR-B1
+ CRTISO
ZEP
a-Carotene
Astaxanthin
CRTO
Mann et al. (2000) Nature Biotechnol. 18:888
An hAT-tye transposon in Psy1 (rArlecchino) Metabolic engineering of astaxanthin biosynthesis
WT 35S:CRTO
Metabolic engineering of astaxanthin in tomato
Nogueira et al. (2017) Engineering of tomato for the sustainable
production of ketocarotenoids and its evaluation in aquaculture feed. Proc.Natl.Acad.Sci.U.S.A. 114:10876-10881.
Metabolic engineering of β-carotene
PDS
CRTISO
GGDP
Phytoene
Lycopene
Prolycopene
ZDS
PSY1
tri-cis-ζ-Carotene
di-cis-ζ-Carotene
Neurosporene
ZDS
ZISO
β-Carotene
PSY2
LCY-B
CRTI
Tobacco (Misawa et al. 1993)
Carrot (Hauptmann et al. 1997)
Rice (Ye et al. 2000)
Tomato (Romer et al. 2000)
Potato (Diretto et al. 2007)
Canola (Ravanello et al. 2003)
Maize (Aluru et al. 2008 , Naqvi et al. 2009)
Wheat (Cong et al. 2009, Wang et al. 2014)
Source: Golden Rice Humanitarian Board
Golden Rice
CYC-B
• Phytoene+Phytofluene: Accumulates in the skin where it absorbing UV rays; Considered a strong anti-inflammatory agent; antioxidant and fights free radicals.
• Lycopene: Promotes healthy, resilient skin; supports the cardiovascular system, implicated in reduced prostate cancer.
• α-carotene: Fights the proliferation of free radicals, may inhibit the development of certain cancers.
• β-carotene: Provitamin A (converts to vitamin A in the body); Supports a healthy immune system; reduce certain cancers.
• β-cryptoxanthin: Reduce the risk of lung cancer and colon cancer by 30%; reduces risk for rheumatoid arthritis by 41%; bone formation; provitamin A.
• Zeaxanthin: Strong antioxidant; found in the retina and lens of the eye where it protects against age-related macular degeneration (AMD).
• Lutein: Strong antioxidant; found in the retina and lens of the eye where it protects against AMD; important for brain development in infants and cognitive functions.
• Astaxanthin: Supports a healthy cardiovascular system; may be able to temper the effects of age-related diseases.
Health benefits of carotenoids
Nutritional and health Engelmann, N.J., Clinton, S.K. and Erdman, J.W., Jr. (2011) Nutritional aspects of phytoene and phytofluene, carotenoid precursors to lycopene. Adv. Nutr. (Bethesda. ) 2, 51-61.
Melendez-Martinez, A.J., Mapelli-Brahm, P., Benitez-Gonzalez, A. and Stinco, C.M. (2015) A comprehensive review on the colourless carotenoids phytoene and phytofluene. Arch. Biochem. Biophys. 572:188-200
Industrial Cosmetics, IBR
Phytoene & phytofluene (P&P) as valuable
natural products
Phytoene= 15-cis-7,8,11,12,7',8',11',12'-Octahydro-ψ,ψ-carotene
Phytofluene= 15-cis-7,8,11,12,7',8'-Hexahydro-ψ,ψ-carotene
Phytoene
Phytofluene
Tomato is the major dietary source of P&P
Ripe* Overripe** Overripe**
+3 h air
0%
20%
40%
60%
80%
100%
other carotenoids P&P
* Breaker + 7 days
**Breaker + 22 days
PDS
CRTISO
phytoene
lycopene
prolycopene
ZDS
tri-cis-ζ-carotene
di-cis-ζ-carotene
ZISO
β-carotene
β-xanthophylls
LCYB, CYCB
HYDs
LCYB, LCYE
α-carotene
HYDs
lutein
IPP
Fruits of zeta accumulate P&P
PDS
CRTISO
phytoene
lycopene
prolycopene
ZDS
tri-cis-ζ-carotene
di-cis-ζ-carotene
ZISO
β-carotene
β-xanthophylls
LCYB, CYCB
HYDs
LCYB, LCYE
α-carotene
HYDs
lutein
Co
nce
ntr
atio
n [
μg
/g f
resh
we
igh
t]
zeta
T
0
50
100
150
200
250
300
350
WT(M82)
zeta P&P
Phytoene
Phytofluene
ζ-carotene
Lycopene
Other
Varda Mann, Chen Amsalem
Zeta (z2083)
Breeding of high P&P tomato varieties
PDS
CRTISO
phytoene
lycopene
prolycopene
ZDS
tri-cis-ζ-carotene
di-cis-ζ-carotene
ZISO
β-carotene
β-xanthophylls
LCYB, CYCB
HYDs
LCYB, LCYE
α-carotene
HYDs
lutein
Co
nce
ntr
atio
n [
μg
/g f
resh
we
igh
t]
zeta
T
0
50
100
150
200
250
300
350
WT(M82)
zeta P&P
Phytoene
Phytofluene
ζ-carotene
Lycopene
Other
Zeta+hp+QTLs
Amit Koch, Dani Zamir
P&P accumulate in plastoglobules
Zeta1-1
M82 (wt)
Field trials of P&P tomatoes
Total
carotenoids [μg/g fresh weight]
Percent P&P
All lines 183.8±61.3 64.3±3.5
Top seven 225.8±40.4 65.4±0.8
Top three lines 258.0±41.3 65.9±0.7
Field trial 2015
Total
carotenoids [μg/g fresh weight]
Percent P&P
All lines 195.0±72.4 60.6±3.6
Top four lines 311.2±17.9 64.2±2.3
Field trial 2016
Amit Koch, Dani Zamir
Achievements of P&P
1. New tomato varieties with high concentration of P&P have been developed.
2. High-yield processed tomato varieties with total carotenoid concentration of >250 μg/g fresh weight comprising of >70% P&P were achieved through genetic breeding. This is the highest P&P content ever recorded in plants.
3. The new P&P varieties have been commercialized to a tomato extraction company for the production of oleoresin extract with high concentration of P&P. The product is available for commercial uses.
4. Molecular and cellular mechanisms underlying the phenomenon of P&P accumulation have been demonstrated.
• Phytoene+Phytofluene: Accumulates in the skin where it absorbing UV rays; Considered a strong anti-inflammatory agent; antioxidant and fights free radicals.
• Lycopene: Promotes healthy, resilient skin; supports the cardiovascular system, implicated in reduced prostate cancer.
• α-carotene: Fights the proliferation of free radicals, may inhibit the development of certain cancers.
• β-carotene: Provitamin A (converts to vitamin A in the body); Supports a healthy immune system; reduce certain cancers.
• β-cryptoxanthin: Reduce the risk of lung cancer and colon cancer by 30%; reduces risk for rheumatoid arthritis by 41%; bone formation; provitamin A.
• Zeaxanthin: Strong antioxidant; found in the retina and lens of the eye where it protects against age-related macular degeneration (AMD).
• Lutein: Strong antioxidant; found in the retina and lens of the eye where it protects against AMD; important for brain development in infants and cognitive functions.
• Astaxanthin: Supports a healthy cardiovascular system; may be able to temper the effects of age-related diseases.
Health benefits of carotenoids
Age-related macular degeneration (AMD)
By 2040, the number of individuals in Europe with early AMD will
range between 14.9 and 21.5 million, and for late AMD between 3.9
and 4.8 million. Colijn et al. Ophthalmology. 2017, 124:1753-1763.
0
10
20
30
40
50
60
Car
ote
no
ids
(μg
/gFW
)
carotenoid content
Phytofluene
Phytoene
β-carotene
Lycopen
zeaxanthin
lutein
Tomato fruit contains negligible amounts
of xanthophylls
Tomato variety M82 (field-grown)
PDS
CRTISO
phytoene
lycopene
ZDS
ZISO
β-carotene
LCYB/CYCB* LCYB+LCYE
α-carotene
HYDe+HYDb
lutein
HYDb
zeaxanthin
violaxanthin
neoxanthin
ZEP
NSY+NXD1
Carotenoids in tomato fruit
Carotenoid concentration in fruit
β-cryptoxanthin HYDb
0
20
40
60
80
100
120
140
160
WT *6998
Caro
tenoid
concentr
atio
n [
μg/g
FW
] xanthophylls
β-carotene
γ-carotene
lycopene
prolycopene
neurosporene
ζ-carotene
phytofluene
phytoene
PDS
CRTISO
phytoene
lycopene
ZDS
ZISO
β-carotene
LCYB/CYCB* LCYB+LCYE
α-carotene
HYDe+HYDb
lutein
HYDb
zeaxanthin
violaxanthin
neoxanthin
ZEP
NSY+NXD1
Carotenoids in mutant High-Beta B6998
Carotenoid concentration in fruit
β-cryptoxanthin HYDb
0
20
40
60
80
100
120
140
160
WT *6998
Caro
tenoid
concentr
atio
n [
μg/g
FW
] xanthophylls
β-carotene
γ-carotene
lycopene
prolycopene
neurosporene
ζ-carotene
phytofluene
phytoene
B6998
PDS
CRTISO
phytoene
lycopene
ZDS
ZISO
β-carotene
LCYB+LCYE
α-carotene
HYDe+HYDb
lutein
HYDb
zeaxanthin
violaxanthin
neoxanthin
ZEP
NSY+NXD1
Combining hp3 with High-Beta B6998
hp3
β-cryptoxanthin HYDb
0
10
20
30
40
50
60
6998 HP3 hp3 x 6998 M82
(Car
ote
no
ids
(μg
/g F
W)
Phytofluene
Phytoene
β-carotene
Lycopen
zeaxanthin
lutein
0
10
20
30
40
6998 HP3 hp3 x 6998 M82
Car
ote
no
ids
(μg
/g F
W)
β-carotene
zeaxanthin
LCYB/CYCB* B6998
Total carotenoids in fruit
β-carotene and zeaxanthin in fruit
(gf) (sg/z)
Stay-green mutations in tomato
0
10
20
30
40
50
M82 6998 hp69 hp69gf
)C
aro
ten
oid
s (μ
g/g
FW
Total carotenoids Phytofluene
Phytoene
β-carotene
Lycopen
β-cryptoxanthin
zeaxanthin
lutein
0
5
10
15
20
25
30
M82 6998 hp69 hp69gf
)C
aro
ten
oid
s (μ
g/g
FW
β-carotene and xanthophylls
zeaxanthinβ-cryptoxanthin β-carotene
WT x x/y x/y/z
Zeaxanthin reached in the triple mutant x/y/z 15 μg/gFW, 36% of total carotenoids
WT x x/y x/y/z
Metabolic engineering of of β‐cryptoxanthin &
zeaxanthin in tomato
Phenotype of the triple mutant B6998 /hp3/sgz
0
10
20
30
40
50
M82 6998 hp69 hp69gf
)C
aro
ten
oid
s (μ
g/g
FW
Total carotenoids Phytofluene
Phytoene
β-carotene
Lycopen
β-cryptoxanthin
zeaxanthin
lutein
0
5
10
15
20
25
30
M82 6998 hp69 hp69gf
)C
aro
ten
oid
s (μ
g/g
FW
β-carotene and xanthophylls
zeaxanthin
β-cryptoxanthin
β-carotene
WT B6998 B6998 /hp3 B6998 /hp3/gf WT B6998 B6998 /hp3 B6998 /hp3/gf
Zeaxanthin reached 15 μg/gFW, 36% of total carotenoids
0
10
20
30
40
50
60
70
80
WT 1905-3other zeaxanthin β-crypto
β-carotene Lycopene P&P
Car
ote
no
ids
[μg
/g F
W]
Phenotype of the mutant B6998 /hp3/sgz/hp1/QTLs
Acknowledgements
Orly Dery
Uri Karniel
Daniel Limoey
Tal Makov
Varda Mann
Avraham Pash
Omer Perach
Marc Simanovich
Research support
Collaboration
Dani Zamir
Itay Zemach
Amit Koch
Israel Science Foundation grant 850/13
EC FP7 KBBE programme DISCO, project 613513.