L9-10: Genetic engineering of biosynthetic

pathways in transgenic plants

L9-12: Genetic engineering of biosynthetic
pathways in transgenic plants
Gene silencing in plants
Discovery
Applications
Reduction of lignin, ethylene, polyphenol oxidase

Improvement of protein composition in transgenic plants

Improvement of vitamin content
Other examples of biofortified food
 Gene silencing in plants

Gene structure mRNA

Inverted
siRNA
duplication
dsRNA
small interfering RNA

3
 RNAi

The discovery

5’44”

https://www.youtube.com/watch?v=H5udFjWDM3E&feature=related
15’ full version https://www.youtube.com/watch?v=Vh3-NHdjnyQ 4
 Principle of RNAi

5’

http://www.nature.com/focus/rnai/animations/index.html 5
 What are small RNAs?
(e.g. siRNA, miRNA#)
#miRNA=microRNA

• pool of 21 to 24 nt RNAs that

generally function in gene silencing

• contribute to post-transcriptional
AAAAA
gene silencing by affecting mRNA
translation or stability

• contribute to transcriptional gene

silencing through epigenetic RNA Pol

modifications to chromatin:
Histone modification, DNA methylation
siRNAs direct and promote DNA methylation
DNA methylation inhibits transcription
Methylated DNA form complexes with proteins involved in deacetylation of histones
When histones are deacetylated, transcription is inhibited

6
 Transcriptional gene silencing
Small RNAs can initiate gene silencing through
covalent modifications of the DNA or its associated
histone proteins, interfering with transcription

Transcription
Histone
proteins

DNA
Silencing

siRNAs direct and promote DNA methylation

DNA methylation inhibits transcription
Methylated DNA form complexes with proteins involved in deacetylation of histones
When histones are deacetylated, transcription is inhibited

7
 siRNAs can target DNA for silencing by
cytosine methylation or by histone modification
The precise mechanisms by which siRNAs
NH2 NH2
CH3 target DNA for silencing are not known, but
N N
involve the action of two plant-specific RNA-
O
N
O
N polymerase complexes, RNA Polymerase IV
(Pol IV) and RNA Polymerase V (Pol V)
~ ~
cytosine 5-methylcytosine
DNA
methyltransferase
DNA can be covalently
modified by cytosine
methylation, carried out by
DNA methyltransferases

siRNAs direct and promote DNA methylation

DNA methylation inhibits transcription Histone modification DNA methylation
Methylated DNA form complexes with proteins involved in deacetylation of histones
When histones are deacetylated, transcription is inhibited

8
 The core of RNA silencing:
Dicers and Argonautes
RNA silencing uses a set of dsRNA
core reactions in which
double-stranded RNA (DCL)

(dsRNA) is processed by sRNAs

Dicer or DiCer-Like proteins
AGO
into short RNA duplexes.

These small RNAs AGO

subsequently associate with

ARGONAUTE proteins to
Silencing
confer silencing.

9
 Dicer and Dicer-like proteins
In siRNA and miRNA biogenesis, Dicer or
Dicer-like (DCL) proteins cleave long dsRNA
or fold back (hairpin) RNA into ~ 21–25 nt
fragments.

Dicer’s structure allows it to measure the RNA it

is cleaving. Like a cook who “dices” a carrot,
Dicer chops RNA into uniformly-sized pieces.

From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006) Structural basis for double-
stranded RNA processing by Dicer. Science 311: 195 -198. Reprinted with permission from AAAS. Photo credit: Heidi

10
 Argonaute proteins
ARGONAUTE proteins bind small
RNAs and their targets.

The Arabidopsis ago1 mutant and

the octopus Argonauta argo
ARGONAUTE proteins are named
after the argonaute1 mutant of
Arabidopsis; ago1 has thin radial
leaves and was named for the
octopus Argonauta which it
resembles.
Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998) AGO1 defines a
novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L.
(2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – 1437. with permission of AAAS.

11
Discovery of induced silencing
In the 1980s, scientists developed
methods for introducing genes into
plant genomes, using the bacterium Plant Cell

Agrobacterium tumefaciens. The

introduced genes are called Nucleus
transgenes.
DNA

Agrobacterium
tumefaciens on the
surface of a plant cell.

Photo credits: Martha Hawes, University of Arizona.

12
 Silencing of transgenes
• Transgenes introduced into plants are frequently
silenced by the siRNA pathway
• Silencing can be triggered by:
• Very high expression levels
• dsRNA derived from transgene
• Aberrant RNAs encoded by transgenes
• Transgenes are silenced transcriptionally and
post-transcriptionally

13
Initial experiments

Experiments to modify flower

color in petunia gave early
evidence of RNA silencing

14
 Manipulation of chalcone synthase
expression to modify pigmentation
Phenylalanine Cinnamic acid Coumaric acid
PAL C4H

Chalcone synthase
(CHS)

Sinapate
Chalcone

Wild-type petunia
producing purple
anthocyanin Chalcone synthase (CHS) is
pigments the enzyme at the start of the
biosynthetic pathway for
anthocyanins Anthocyanins
https://cdn6.bigcommerce.com/s-q83qdckkjh/images/stencil/1024x1024/products/179/508/Wild_Petunia__83218.1499031317.jpg?c=2
Photo credit Richard Jorgensen; Aksamit-Stachurska et al. (2008) BMC Biotechnology 8: 25.

15
 Expectation – sense RNA production
would enhance pigmentation...

Endogenous gene Protein translated

mRNA
PRO ORF
mRNA

Transgene Extra protein translated

Sense construct: Sense RNA mRNA
PRO ORF
mRNA

16
 ..and antisense RNA production
would block pigmentation
Endogenous gene Protein translated
mRNA
PRO ORF
mRNA

Transgene Extra protein translated

Sense construct: Sense RNA mRNA
PRO ORF
mRNA

Transgene
Sense-antisense duplex forms and
Antisense construct: Antisense prohibits translation
ORF RNA
PRO

17
 Results: Both antisense and sense gene
constructs can inhibit pigment production

Plants carrying CHS transgene

CaMV 35S pro : CHS OR CaMV 35S pro : CHS

Sense Antisense

Photo credit Richard Jorgensen

18
 Silenced tissues do not express
endogenous or introduced CHS

This phenomenon, in which

both the introduced gene
and the endogenous gene
are silenced, has been
called “co-suppression”.

Purple flowers White flowers

Napoli, C., Lemieux, C., and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in
trans. Plant Cell 2: 279–289.

19
 Co-suppression is a consequence of
siRNA production
Wild-type Protein translated
mRNA
PRO ORF
mRNA
Endogenous gene

Co-suppressed transgenic Co-suppression

Sense RNA
PRO ORF
Sense construct siRNA
produced
PRO ORF mRNA
Endogenous gene
De Paoli, E., Dorantes-Acosta, A., Zhai, J., Accerbi, M., Jeong, D.-H., Park, S., Meyers, B.C., Jorgensen, R.A., and Green, P.J. (2009). Distinct extremely abundant siRNAs
associated with cosuppression in petunia. RNA 15: 1965–1970.

20
 Studies of C. elegans showed double-
stranded RNA is the strongest trigger
for gene silencing
Sense, antisense or
double-stranded RNAs
Sense RNA Double-stranded RNA
homologous to the unc- Anti-sense RNA
22 gene were
introduced into worms.
Silencing of unc-22
causes loss of muscle
No effect No effect Uncoordinated twitching
control – hence its
name, “uncoordinated”.

Derived The Nobel Committee based on Fire, A. et al., (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature 391: 806-811.

21
What is the role of
siRNA?
 siRNA is the genomic defender
siRNAs protect the genome by
• Suppressing invading viruses
• Silencing sources of aberrant transcripts
• Silencing transposons and repetitive
elements
• siRNAs also maintain some genes in
an epigenetically silent state

Reprinted by permission from Macmillan Publishers, Ltd: Nature. Lam, E., Kato, N., and Lawton, M. (2001) Programmed cell death, mitochondria and the plant
hypersensitive response. Nature 411: 848-853. Copyright 2001.

23
 Viral induced gene silencing -
overview
Virus-encoded
RNA-dependent
RNA polymerase

Viral dsRNA
Viral ssRNA

Most plant viruses are Double-stranded RNA is

RNA viruses that
replicate through a
double-stranded RNA
intermediate
cleaved by DCL to
produce siRNA which AGO
associates with AGO to
silence virus replication
and expression

AGO

24
 Plants can recover from viral
infection and become resistant
YOUNGEST Younger leaves
produced on a virus-
infected plant can be
symptom-free,
indicating that the plant
has recovered from the
OLDEST
infection
Inoculate with
virus

25
 Plants can recover from viral
infection and become resistant
OLDEST YOUNGEST

Leaf inoculated No symptoms: Leaf inoculated

with virus with virus
recovery
Virus-induced No symptoms:
cell death resistance
From Ratcliff, F., Henderson, B.D., and Baulcombe, D.C. (1997) A similarity between viral and gene silencing in plants. Science 276: 1558–1560.
Reprinted with permission from AAAS.

26
 Plants can recover from viral
infection and become resistant
YOUNGEST Inoculate with These leaves are
virus
also resistant to
subsequent
infection by the
same virus
OLDEST

Inoculate with
virus

27
Virus infection causes systemic
siRNA accumulation?

Distal leaf

Inoculated leaf

28
 How do we know RNA silencing spread
systemically?
UV phenotypes of N. benthamiana

GFP

Under UV light, wild- A plant expressing

type leaves fluoresce GFP fluoresces
red, from chlorophyll green under UV light
in the chloroplasts

Reprinted with permission from Kalantidis, K., Schumacher, H.T., Alexiadis, T., and Helm, J.M. (2008) RNA silencing movement in plants. Biol. Cell 100: 13–26; (c) the Biochemical Society.

29
Examination of RNA silencing

GFP
GFP

To examine gene
silencing, GFP-encoding
inverted-repeat (IR) DNA
is introduced into the
GFP-expressing cells

When GFP is silenced, the red chlorophyll

fluorescence becomes visible.
30
Silencing can spread locally
Often the silencing
spreads over up to
15 cells, probably by
diffusion of the
silencing signal
through the
plasmodesmata

Cell
Wall



Plasmodesmata are regulated

connections between plant cells
Reprinted from Zambryski, P. (2008) Plasmodesmata. Curr. Biol. 18: R324-325 with permission from Elsevier. TEM image credit BSA Photo by Katherine Esau;
31
 Results showing the systemic
spread through the phloem
2 2
3 3
Lower leaf
infiltrated earlier

Anti-GFP
1 1
showing the
progression
of GFP-
4
4 silencing

Reprinted by permission from Macmillan Publishers, Ltd: Nature Copyright 1997. Voinnet, O., and Baulcombe, D. (1997) Systemic silencing in gene silencing. Nature 389: 553.

32
 siRNA is a defensive mechanism
to viral disease
WT Arabidopsis dcl2-dcl4 double mutant
inoculated with TRV inoculated with TRV

Tobacco Rattle Virus (TRV) Mutants deficient in Dicer

silencing in wild-type activity are unable to
Arabidopsis plants prevents suppress viral infection
disease symptoms

From Deleris, A., Gallego-Bartolome, J., Bao, J., Kasschau, K., Carrington, J.C., and Voinnet, O. (2006) Hierarchical action and inhibition of plant dicer-like proteins in antiviral defense. Science 313: 68–71. Reprinted
with permission from AAAS.

33
 Viral-induced gene silencing
summary
• RNA-mediated gene silencing is an important
tool in plant defense against pathogens
• siRNAs interfere with viral replication
• siRNAs act systemically to aid in host plant
recovery and resistance

Viral infection Detection of dsRNA RNA interference

Images extracted from https://www.youtube.com/watch?v=Vh3-NHdjnyQ
34
 Recognition & initiation of RNAi
• Healthy plants do not contain dsRNA
• Most plant viruses have ssRNA genomes
• replicate in the cytoplasm using own RDRP to
produce both sense (plus-strand) and antisense
(minus-strand) RNA
• produces sufficient stretches of dsRNA and attracts
Dicer or DCL

35
Gene silencing as an adaptive defence against viruses Nature 411, 834 - 842 (14 June 2001)
A model for RNAi in plant cells
Most plant viruses have ssRNA
genomes and replicate using
RDRP and dsRNA
IR transgene in nucleus RNA virus
(red-white DNA indicates inverted repeats;
yellow arrow indicates direction of transcription) in cytosol

Methyltransferase

RNA-directed
methylation

1. dsRNA produced from transgene in nucleus or

from viral RNA genome in cytosol
2. Dicer or DCL proteins in cytosol bind dsRNA at ends
3. dsRNA cleaved to 21 nucleotide complexes
(i) spread systemically to other parts of plant
(ii) transported into nucleus to direct DNA methylation (transcriptional gene silencing)
4. DCL complex acted as guide for cleavage of homologous ssRNA
Waterhouse et al. Nature 411, 834-842 (14 June 2001)
(post-transcriptional gene silencing) 36
 Tobacco protection against potato virus Y
(PVY) by RNAi:
susceptibility, immunity and resistance
Wildtype tobacco
Virus-challenged
challenged with
transgenic plant which
PVY
express hairpin RNA
Note chlorotic
derived from PVY
mosaic on leaves
No symptoms: immune
throughout plant

As plant grows, As virus attempts to

leaves show fewer spread, signal is
yellow patches emitted that
(yellow patches forewarns distal
contain detectable cells, resist and
levels of virus) restrict the virus
c. Whole leaf, and d, close-up, from a PVY-challenged
transgenic plant showing resistance/recovery symptoms
37
Waterhouse et al. Nature 411, 834-842 (14 June 2001)
 siRNAs - summary
The siRNA pathway silences foreign DNA, transposons and
repetitive elements
In plants, siRNAs are produced by the action of Dicer-like
proteins slicing dsRNA into 21-24 nt siRNAs
The siRNAs associate with AGO proteins and form silencing
complexes
The silencing complexes can act post-transcriptionally on
RNA targets, cleaving them or interfering with translation
The silencing complexes can also act on chromatin, silencing
their targets by DNA methylation or histone modification

38
 Conclusions
Small RNAs contribute to the regulation and defence
of the genome, and confer silencing specificity
through base-pairing

siRNA targets include repetitive-rich heterochromatin,

transposons, viruses or other pathogens

39
 Applications of siRNA technology
In plants, siRNA or
miRNA-forming DNA Gene silencing Pest Control
can be introduced can eliminate
stably into the genome allergens from
to selectively silence peanuts
one or more genes

Control, infected by RNAi-inducing – no

parasitic nematode infection

Plants expressing dsRNA

Gene silencing can corresponding to insect or
remove toxic nematode genes are resistant
compounds from cotton to these pests! When plant
seed so they can be ingested, the dsRNA induces
used as a food source gene silencing in pest

Huang, G., Allen, R., Davis, E.L., Baum, T.J., and Hussey, R.S. (2006) Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc.
Natl. Acad. Sci. USA 103: 14302–14306.
40
 Use of RNAi in metabolic engineering of plants
Mansoor et al., 2006. TPS 11:559

*Details in next PPTs

Trait Target gene Host Potential benefit

Enzymatic browning Polyphenyl oxidase Potato Extended storage

life

*Reduced caffeine MXMT Coffee Decaffeinated coffee

7- N -methylxanthine methyltransferase

*Increased carotenoid DET1 Tomato Health benefits

and flavonoid DE-ETIOLATED1

Reduced ethylene ACC oxidase Tomato Longer shelf life

http://www.ncbi.nlm.nih.gov/pubmed/15604660 Ogita S et al, Plant Mol Biol 54:931-941

Application of RNAi in caffeine biosynthesis with potential for decaffeinated varieties

Caffeine biosynthetic pathway in coffee plants involve 3 distinct N-methyltransferases:

1. xanthosine methyltransferase (XMT),
2. 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and
3. 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase)

cDNAs (XMT , MXMT and DXMT , respectively) cloned and tested

To regulate caffeine biosynthesis in planta , suppressed expression of MXMT1 by RNAi

Established protocol for efficient somatic embryogenesis of Coffea & Agrobacterium

transformation

RNAi transgenic lines showed reduction in MXMT mRNA in comparison to control

Plantlets exhibited reduction of theobromine and caffeine contents (30% & 50% of control)

Method can be practically applied to produce decaffeinated coffee plants

http://www.ncbi.nlm.nih.gov/pubmed/15951803 Davuluri et al Nat Biotechnol. 2005, 23:890-895

Fruit-specific RNAi-mediated suppression of DET1 enhances

carotenoid and flavonoid content in tomatoes

DET1 = DE-ETIOLATED1, photomorphogenesis regulatory protein that negatively regulates light response

Tomatoes, dietary source of carotenoids* and flavonoids* beneficial for human health
Overexpression of genes encoding biosynthetic enzymes or transcription factors have
resulted in tomatoes with improved carotenoid or flavonoid content, but never with both

Strategy:
Increase tomato fruit nutritional value by suppressing an endogenous
photomorphogenesis regulatory gene DET1 using fruit-specific promoter combined with
RNAi technology
DET1 transcripts specifically degraded in transgenic fruits
Both carotenoid and flavonoid contents increased significantly; other parameters of fruit
quality remained unchanged
Demonstrate manipulation of a plant regulatory gene can simultaneously influence the
production of several phytonutrients generated from independent* biosynthetic
pathways; use of organ-specific gene silencing to improve the nutritional value
A new GM alfalfa with reduced lignin
http://www.nature.com.eproxy2.lib.hku.hk/nbt/journal/v33/n1/box/nbt0115-12_BX1.html
E. Waltz. Nature Biotech 33, 12–13. doi:10.1038/nbt0115-12

• Alfalfa genetically modified to have reduced levels of lignin

• So growers have greater flexibility in timing harvest without sacrificing the quality
of plant
• Food for ruminants, but its lignin increase makes it indigestible
• Increase in alfafa biomass related to lower quality of forage
• Reduce guaiacyl lignin will lower total lignin by 15–20%
• Growers have flexibility to delay harvest by up to a week while maintaining the
quality of the forage
• Alfafa usually harvested at 1 mth

Expt:
Alfafa DNA segments for caffeoyl CoA 3-O-methyltransferase (CCOMT) were
used in RNAi construct
Plants showed lower CCOMT expression and reduced synthesis of guaiacyl lignin

This is the second GM alfalfa trait to be approved in the US

The first was glyphosate-tolerant alfalfa developed by the same companies
 Example: GM Alfalfa
Alfalfa
plants

Alfalfa sprouts

Alfalfa farmed for hay

45
http://study.com/academy/lesson/alfalfa-definition-uses-benefits.html
 Yield vs digestibility
Alfalfa is
usually
harvested
at monthly
intervals

Relative forage yield and quality at different alfalfa growth stages.

Lignin in alfalfa increases as the plant grows and become indigestible.
Increase in alfalfa biomass = lower quality of forage
46
Source: Balancing Yield, Quality and Persistence. Steve Orloff and Dan Putnam 2004 Proceedings CA Alfalfa Symposium
 GM Alfalfa

Caffeoyl-CoA

Caffeoyl CoA 3-O-MethylTransferase

Coniferyl Coniferyl Guaiacyl
Feruloyl-CoA
aldehyde alcohol lignin
F5H

5-hydroxy 5-hydroxy-
coniferyl guaiacyl
alcohol lignin
COMT

Syringyl
F5H: Ferulate 5 Hydroxylase lignin
COMT: Caffeic acid O-methyltransferase 47
 GM Alfalfa
Developed by Monsanto and
Shoreview, Minnesota–based
Caffeoyl-CoA Forage Genetics International
By RNAi
X Caffeoyl CoA 3-O-MethylTransferase
Coniferyl Coniferyl Guaiacyl
Feruloyl-CoA
aldehyde alcohol lignin
F5H
Reduced-lignin trait reduce
guaiacyl lignin reducing total 5-hydroxy 5-hydroxy-
lignin by about 15-20% coniferyl guaiacyl
Give farmers the flexibility to alcohol lignin
delay harvest by up to a week COMT
while maintaining the quality
Syringyl
lignin
F5H: Ferulate 5 Hydroxylase
COMT: Caffeic acid O-methyltransferase This is the second GM alfalfa trait to be approved in the US. 48
The first was glyphosate-tolerant alfalfa developed by the same companies
Application: Post-harvest manipulation

After harvesting,
fruits soften, ripen,
and eventually rot.

These processes make the fruit less

appealing and affect the nutritional
qualities.
49
http://static0.7sur7.be/static/photo/2013/11/11/12/20130620132046/media_l_5926092.jpg https://i.guim.co.uk/img/static/sys-images/Guardian/Pix/pictures/2015/9/10/1441899437453/Food-Chains-
https://news.plantsciences.ucdavis.edu/wp-content/uploads/2015/07/15.07.24-tomato-harvester-STAR- 009.jpg?width=300&quality=85&auto=format&fit=max&s=5788b97989651fcd20556ee48b5d8077
NTD-Tomato-Harvester-e1436906748749-680x418.jpg http://fb.ru/misc/i/gallery/29821/1656543.jpg

First GM Food: Flavr Savr tomato

Supermarket wild-type tomatoes not as tasty as
vine-ripened tomatoes (picked when green;
then exposed to ethylene to induce redness)
• Calgene-engineered tomato (ripen on vine but
transported without bruising)
• “Softening” was silenced
• stay on vine few days longer
• allows sugars transported to fruit: taste better
• Firm texture persist; longer shelf-life

50
 How the Flavr Savr works?
Use of antisense RNA
Tomato fruit softening gene antisense
polygalacturonase Polygalacturonase construct

DNA

Antisense
PG mRNA

mRNA

dsRNA triggers
degradation
Inactivated RNA
51
https://thehealthreportertv.files.wordpress.com/2013/07/plu-label_gmo.jpg
 Another silencing approach?
Antisense ACC synthase
Oxidation
MACC products
Malonyl
Transferase
Fruit
SAM ACC C2H4
ACC ACC Activation of ripening
Synthase Oxidase ripening by
ethylene
Feedback

Pathway for ethylene synthesis and metabolism

SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylic acid; MACC, malonly-ACC; Feedback,

positive & negative effect of ethylene on its own biosynthesis

52
Oeller P.W. et al, Science 254: 437-439
 Anti-ACC synthase
+1

PstI SalI BamHI EcoRI SacI EcoRI

pPO35
35S Lc-tACC2 NOS
0.5 kb

Lycopersicon esculentum ACC2 cDNA

inserted in reverse orientation in pBI101
and used to transform tomato

The pPO35 plasmid was transferred from Escherichia coli DH5α to Agrobacterium
tumefaciens LBA4404 by triparental mating with E. coli HB101 harbouring pRK2013

53
 Production of ethylene in tomato fruits
Control 1 Wildtype: Air

Control 2 Wildtype: Propylene

(propylene to induce ripening)

Transgenic 1 Air

Transgenic 2 Propylene

No production of ethylene
from transgenic tomato

Days after pollination

54
Oeller P.W. et al, 1991. Science 254: 437-439
Treatment with ethylene reverse
antisense phenotype

Control: 45-d-old fruit tested

Transgenic 1 Air
Transgenic 2 Ethylene

Adding of ethylene allows

ripening to progress

45d
old
fruit Days after pollination

There is a respiratory burst during the ripening process 55

 What is the difference? C3H6: propylene
induces ripening

Control produce ethylene

48-50 days after pollination,
then ripen after 10 more
days
Antisense fruits do not
produce ethylene, fail to
ripen (only turns orange
never red and soft)

Propylene treatment can

reverses antisense
phenotype

56
Oeller P.W. et al, 1991. Science 254: 437-439
 Ethylene treatment restores ripening
in antisense tomatoes

Air

Effect of C2H4 treatment on the antisense phenotype

Mature green fruits from control and homozygous antisense plants were harvested 49 days after pollination
and treated with air for 15 days or with 10 µl of C2H4 per liter of air for 1, 2, or 15 days and then returned to
air. Fruits were photographed on day 16 after harvesting.
57
Oeller P.W. et al, 1991. Science 254: 437-439
 Any other silencing approach?
Antisense ACC oxidase
Oxidation
MACC products
Malonyl
Transferase
Fruit
SAM ACC C2H4
ACC ACC Activation of ripening
Synthase Oxidase ripening by
ethylene

Also known as Ethylene Feedback

Forming Enzyme (EFE)
Pathway for ethylene synthesis and metabolism

SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylic acid; MACC, malonly-ACC; Feedback,

positive & negative effect of ethylene on its own biosynthesis.

58
Oeller P.W. et al, 1991. Science 254: 437-439
Ethylene production in EFE-antisense
7 ▲ wildtype, attached
Ethylene evolution (nl/g per hour)
6 ■ wildtype, detached
∆ EFE-antisense,
5 attached
□ EFE-antisense,
4 detached

3
Ethylene
2 production is
highly reduced in
1 antisense plants

0
Mature breaker +2 d +4 d +6 d +28 d
Green Tomato fruit ripening stage

59
Picton et al., 1993. Plant J. 3: 469-481 pBASC: 35S-[anti-ACC oxidase]-35Sterm
 Phenotypic changes in EFE-antisense
fruit ripened on the plant

Wildtype

EFE-antisense

Mature green fruit Fruit ripened on Ripened, detached &

the plant for 4 stored for a further 8
weeks following weeks at 25°C
60
Picton et al., 1993. Plant J. 3: 469-481 colour change
 Phenotypic changes in EFE-antisense
fruit off the plant
Air

Wildtype
EFE-antisense Air

Air + C2H4

Days after colour change 28 14 6 Stored for 7 days 61

Picton et al., 1993. Plant J. 3: 469-481
 EFE-antisense inhibits ripening of
cantaloupe melon fruits

HindIII
EcoRV

EcoRV
BglII

BglII
npt II CaMV 35S Anti-EFE term
LB RB
Cantaloupe Charentais melons harvested 38 days
after pollination and stored at 25°C for 10 days

Ethylene production < 1% of control fruit

Ripening process is blocked on and off

the vine

Antisense phenotype can be reversed

by exogenous ethylene treatment

Wildtype Anti-ACC oxidase

62
Ayub R et. al., Nat Biotech. 1996 Jul; 14(7):828-829.
 More examples on gene silencing:
anti-polyphenol oxidase
• Injury during mechanical harvesting of
potato causes discoloured patches
• This ‘browning’ is due to formation of
melanins – oxidation of polyphenols
• Reaction of
polyphenol oxidase
PPO

Bush, Biochem Edu 27, 171-173

https://gmwatch.org/images/banners/Unwanted-GM-Potato-710x300.jpg
63
https://amp.businessinsider.com/images/550cdb666da811f30db5030a-640-480.jpg
 Cloning of the PPO cDNA

Sense & antisense PPO sequences

3 different promoters were used:

CaMV 35S

Potato specific promoters

Granule Bound Starch Synthase
Patatin

Selection cassette Expression cassette

2 varieties of potato transformed
Bachem et. al., 1994. Nat Biotech 12: 1101-1105. 64
Effect of ‘silencing’
Potato expressing antisense PPO

CaMV 35S and GBSS promoters better

Bruising phenotype of an untransformed control (left)

and a transgenic line of the variety Diamant (right)

65
Bachem et. al., 1994. Nat Biotech 12: 1101-1105.
Use RNAi to reduce the level of PPO:
White Russet™ potato

40”

24 h time-lapse
66
http://www.innatepotatoes.com/
 Transgenic potato beats cancer?
• 2002 acrylamide forms in some foods during certain types of high-
temperature cooking
• Division of Plant Products and Beverages, Office of Food Safety, in the Center for Food
Safety and Applied Nutrition at the U.S. Food and Drug Administration

• Acrylamide forms in foods from chemical reaction between

asparagine (amino acid) and reducing sugars (eg glucose, fructose)
• This reaction is part of the Maillard reaction, which leads to colour, flavour, and
aroma changes in cooked foods

• Acrylamide formation occurs primarily in plant-based foods, notably

potato products such as French fries and potato chips

67
Mottram et al., 2002. Nature 419,448-449 Stadler et al., 2002 Nature 419,449.
 Transgenic potato
+ sugar Acrylamide
Asparagine
synthetase
(ASN1)
Aspartate + Glutamine Asparagine + Glutamate

ATP AMP + PPi

J.R. Simplot Company developed potatoes:

Use RNAi to reduce the level of PPO: White Russet™ potato

Also used RNAi to reduce the level of ASN1 lowers the

formation of acrylamide by 58-72% when potatoes are baked,
fried or roasted
68
http://whiterusset.com/assets/Simplot_White_Russet_Brand_Guidelines.pdf
 USDA approved non-browning
GM fruits and vegetables
• Next-generation GM potato
• Previously bruising and browning make them less
visually pleasing, and 400 million pounds of
potato wasted
• Suppressing ASN1 reduces formation of
acrylamide
• Non-browning apple
• Canadian biotech Okanagan Specialty Fruits
• Uses similar PPO-silencing approach

• Future: lettuce, cherries, avocados, bananas

69
E Waltz (2015) Nature Biotech 33:12–13.
 GM Arctic apple

46”

Approved in 2015 and delivered in late 2017

70
https://www.youtube.com/watch?v=9cIFp4a0ZgQ
 GM Arctic apple

29”

24 h time-lapse
71
https://www.youtube.com/watch?v=g2-BqBZmVd0
 Example Exam Questions

Discuss how tomatoes can be genetically altered to delay fruit ripening. Discuss
the relevant biosynthetic pathway and the procedures/techniques used in
generation of these transgenic tomato lines.
Also describe the tests carried out to confirm the feasibility of this approach.

Discuss how reversible inhibition of fruit ripening can be achieved in transgenic

fruits effecting each of 2 different enzymes.

Briefly describe gene silencing. Illustrate with specific examples its applications in
tomato and potato.

Discuss, with specific examples, how anti-sense constructs can be used in the
inhibition of biochemical pathways in transgenic plants.