Una visione sistematica per
l’ingegneria metabolica
Cosa ha da dire la MCA ai
biotecnologi vegetali?
Ingegneria metabolica
probabilmente efficace
probab. inefficace
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
S
S
S
S
S
S
S
S
S
+A
A
A
A
+
A
A
A
A
A
TF
+
B
B
B
B E
B
+
B
B
B
B
Y
Z
W
C
C Q
C F
C
P
P
P Q
P
ATP
Flux change: an asymmetric problem
P
C
C
C
C
P
P
P
P
X
Inattivazione di enzimi
Spegnimento, parziale o totale, di geni tramite:
(2)
S
- Knock out
Glucosinolati (Cyt. P450)
- Antisenso
Biosintesi di lignina
-Cosoppressione
Ac. grassi poli-insaturi (desaturasi)
A
B
-RNA interference
Morfina (COR)
Caffeina
Lisina
Domesticazione?
Amido
......
Inactivation of Allergens
and Toxins
Per approfondire: manoscritto con
150 referenze
Piero Morandini
Dept. of Biology
Milan University (Italy)
Outline
The dangers of nature and food
Gene inactivation strategies
Manipulating crops (toxins)
Manipulating crops (allergens)
Transgenic vs. classical approaches
Consequences & conclusions
 Most of the material presented is the work
published by other groups
Nature: a ‘mine field’
Toxic substances abound in nature, both in cultivated and
wild plants
With time we learned how to avoid some (or at least limit
their intake), inactivate others through various processes:
Proper storage
Cooking (e.g. heat inactivation)
Food processing (e.g.: maceration, fermentation)
precise map
metal detector
Knowledge handed through culture and technology (to detect,
avoid & inactivate) are crucial for survival
Technology and knowledge buffer us from the toxic effect of nature
A field trip into the ‘mine field’
Plant
Toxic substance
Effect Dose
Assa foetida
Prenylated coumarins
lethal
Datura stramonium
Iosciamine/scopolamine atropine
lethal
2 ng/ml blood
Tobacco
Nicotine
lethal
20g leaf
Solanum sodomeum
Solasonine, solanidine.
toxic
Ricinus (castor bean)
Ricin / Ricinoleic acid
lethal
1 seed
Tomato
tomatine
Potato
solanine
lethal
3-6 mg/Kg
Cassava
Cyanogenic glucosides
lethal?
Beans
Protease / amylase inhibitors, lectins
toxic
Almond
Cyanogenic glucosides
lethal
Brassicas
Glucosinolates
toxic
20 seeds
Focus mainly on small metabolites (but toxic proteins also abound in nature)
http://www.poppyseedtea.com/ Poppy seed tea can kill you
Solanum tuberosum
http://commons.wikimedia.org/wiki/File:Potato_sprouts.jpg
March 27, 1925 340-341
http://depthofprocessing.blogspot.com/2009
/05/are-potato-peels-nutritious.html
Poisoning and Toxicology Handbook
by Leikin & Paloucek 4th edition,
Informa Health Care, 2007
ISBN 1420044796, 9781420044799
Solanine abounds in green parts, sprouts and diseased potatos
Solasonine
LD50=30 mg/kg
http://kanaya.naist.jp/knapsack_jsp/image.jsp?word=C00002265
Solasonine accumulates in Solanum sodomeum
Datura stramonium
http://www.luciolongo.it/Fotografie%20strane
%20che%20ho%20fatto%20in%20giro.htm
angel's trumpet or devil's weed
* Lazzarini, D. et al. (2006) Food poisoning by Datura stramonium: an unusual
case report. Intern. Emerg. Med. 1:88-90.
* Suk, SH and Kwak, YT (2009) Toxic encephalopathy after taking dried seeds
of Datura stramonium in two elderly subjects. Geriatr. Gerontol. Int. 9:326-328.
* Spina, SP and Taddei, A (2007) Teenagers with Jimson weed (Datura
stramonium) poisoning. Canadian Journal of Emergency Medicine 9:467-468.
* Arnett, A.M. (1995) Jimson Weed (Datura stramonium) Poisoning. Clinical
Toxicology Review 18. (Available at:
http://www.erowid.org/plants/datura/datura_info5.shtml).
Capsicum annuum
peperoncino
www.floralimages.co.uk/pdaturstram1.htm
* Snyman, T., Stewart, M.J. and Steenkamp, V. (2001) A fatal
case of pepper poisoning. Forensic Sci Int. 124, 43-46.
*http://members.tripod.com/prof_anil_aggrawal/poiso010.html
http://www.ubcbotanicalgarden.org/potd/2006/10/capsicum_annuum_cultivars.php
Teenagers with Jimson weed (Datura stramonium) poisoning
A teenager brought a Jimson weed plant to a party after watching youths misusing the plant
on a popular television show. Eight teenagers opened the seed pods, each chewing and
ingesting the seeds from 2 to 3 pods (~100-300 seeds) in combination with alcohol. A 16year-old white male and a 15-year-old female of Asian descent presented to our tertiary
care emergency department (ED) with a severe acute anticholinergic toxidrome after this
ingestion, which was 1-2 hours before presentation. The male patient … presented with
visual hallucinations, disorientation, incomprehensible and nonsensical speech, and dilated
sluggish pupils. The patient was tachycardic (heart rate 120 beats/min), febrile (38°C) and
had a blood pressure of 120/60 mm Hg.
The female patient was very agitated, disoriented, swearing and spitting at ED staff after
ingestion of approximately 100 Jimson weed seeds and vodka. On exam, she had dilated
pupils, sinus tachycardia (heart rate 160 beats/min), tachypnea (40 breaths/min), fever
(37.8°C)…
* 188 human cases over a 6-year period in Texas. 78% of cases were
the result of intentional abuse, largely in teenagers.
* 23 cases were reported in British Columbia over 3 years (2004-2006).
* ingestion was possibly associated with a teen fatality in British
Columbia in 2006.
* The use of Jimson weed can be prevalent in the teenage party
community as a means to achieve an inexpensive euphoria. Jimson
weed seeds are easily purchased online.
Case report: http://www.cjem-online.ca/v9/n6/p467
http://uvalde.tamu.edu/herbarium/final/dain_fr.jpg
Xanthotoxin or 8-methoxypsoralen
Celery contains several natural pesticides
http://en.wikipedia.org/wiki/Psoralen
Psoralens form adducts with DNA upon
exposure to light and therefore cause
photodermatitis (= light dependent
dermatitis)
Giant hogweed
http://nyis.info/plants/Images/GH_burns.jpg
http://nyis.info/plants/Images/GH_plant.jpg
For giant hogweed to affect a person, sap
from a broken stem or crushed leaf, root,
flower or seed must come into contact with
moist skin (perspiration will suffice) with
the skin then being exposed to sunlight.
See also http://www.pathguy.com/sol/12523.jpg
Giant hogweed, like celery, is
a member of the carrot and
parsley family
Two days
and
three days
after contact and moderate light exposure
Image from the author’s body, as punishment for not recognizing the plant in an
orchard while collecting plums (despite all the academic knowledge...)
Colchicine intoxication
http://pandasthumb.org/archives/2010/08/08/
Samarasinghe.Gloriosa_superba.JPG
http://en.wikipedia.org/wiki/File:
Colchicum_speciosum000.jpg
Colchicine is a potent poison: it
blocks microtubule formation
and thereore separation of
chromosomes at cell division.
http://en.wikipedia.org/wiki/Colchicine
Ide et al. (2010) [Case of colchicine intoxication caused by tubers of Gloriosa
superba] [in Japanese] Chudoku Kenkyu. 23:243-5.
Gloriosa superba is one of the poisonous plants growing in Japan. It contains potent
alkaloid such as colchicine …
We report here a case of colchicine intoxication caused by tubers of Gloriosa
superba. A 58-year-old male ingested about 25 g of Gloriosa superba tubers by
mistake. He believed that it was wild yam. He developed abdominal pain, vomiting
and diarrhea 30 minutes after the ingestion of the plant. Forty five hours later he
was taken to the emergency department. Unfortunately he died due to
progressive multiple organ failure about one hour after the admission. It was
two days after Gloriosa superba ingestion.
Kupferschmidt and Campbell (2005) Colchicine Poisoning. A 5-Year European
Poisons Centres Survey. Clinical Toxicology 43:399
Poisoning from ingestion of meadow saffron leaves or from colchicine tablets is a
rare but regular medical emergency.
Number of fatal cases, and the respective number of colchicine tablet and C.
autumnale poisonings during the years 1999–2003.
Reported 355 cases of colchicine poisoning, 15 with a fatal outcome, in the
EU. In 119 cases (34%), ingestion of C. autumnale material was involved (only
1report with no information about the source of colchicine, i.e. plant or tablet). 
an estimate of 1100 cases with 45 fatalities per 5 years.
A matter of dose (threshold)  reduction below a threshold considered safe
Latin name
Toxic substance1
Effect2
Dose3 |
Ferula communis
Prenylated coumarins
lethal
Datura stramonium
Atropine (and other alkaloids)
lethal
100 seeds | 0.1 mg/seed
Nicotiana tabacum
Nicotine
lethal
1 mg/kg
Solanum sodomeum
Solasonine, solanidine
toxic
30 mg/kg | 0.3 mg/g
Ricinus communis
Ricin / Ricinoleic acid
lethal
half a seed
Capsicum spp.
Capsaicin
lethal
Solanum lycopersicum
Tomatine
toxic
Solanum tuberosum
Solanine
lethal
Manihot esculenta
Cyanogenic glucosides
paralysis-stunting
Glycine max
Protease / amylase inhibitors
toxic
Prunus dulcis
Cyanogenic glucosides
lethal
Brassica oleracea
Glucosinolates
lethal-goiter
Gossypium hirsutum
Gossypol
cardio/ hepatotoxic
0.3-3 mg/kg | 10 mg/g
Lathyrus sativus
oxalyl-diaminopropionic acid
neurotoxin /paresis
|
Phaseolus lunatus
Cyanogenic glucosides
lethal
Papaver somniferum
Morphine
lethal
Several species
Cyanogenic glucoside
toxic
Aflatoxins
Cancer
Maize
Fumonisins
Cancer / spina bifida
| 3-6%
| 0-2 mg/g
| 3-6 mg/Kg
| 15-400 mg HCN/kg
20 seeds
| 29 mg/kg
|
1-2 mg/g
0.3-3.2%
| 2-3 mg HCN /kg
100 mg
| 10 mg/g
| 1-8 g HCN /kg
Mycotoxins
Maize, peanut
content4
1-5 mg/kg
The issue of toxic substances in plants is not new nor is gone
Cultivated plants have less toxins than wild relatives (e.g. potato)
What happened? Mutants selected by human & animal tests
Trial and error (or trial and death)
Many crops still produce low levels of toxins (capacity is there!)
Their content may increase by breeding or spontaneously:
e.g. Potato, Celery, Zucchini (courgette)…
http://www.springerlink.com/content/37827612x62xp348/
Bottle gourd
Celery
How to reduce toxic substances in plant?  Target the gene(s)
Gene  RNA  Protein
Toxins are either proteins or are produced through proteins
http://nonsense123.files.wordpress.com/2008/06/bottle-gourd.jpg
Which approaches?
Zucchini Blackjack
http://www.people.cornell.edu/pages/kjc34/distribution.html
http://www.growfruitandveg.co.uk/grapevine/vegging-out/courgettes-doing-my-head_18095.html
Cucurbitacins in Plant Food
Di Jørn Gry, Inge Søborg and Hans Christer
Andersson
Recupera formule di struttura...
Libro su goggle books
Gene inactivation strategies
a) inactivate a protein
(enzyme)
Regulator
b) increase toxin
degradation
c) target a regulator
of the synthesis
Degradation
product
Halkier (2006) Annu. Rev.
Plant Biol. 57:303-33
Tools of the trade
to inactivate (plant) genes
 ‘Classical’ mutation (base change, insertion, deletion…)
 Insertional mutagenesis (transposons or T-DNA)
 RNA mediated (antisense, RNAi, miRNA, hpRNA…)
collectively known as post-transcriptional gene silencing
(PTGS), often involving epigenetic changes
Different methods may end up exactly in the same result
(inactivation of a gene) and the same change at the DNA level
Origin
Advantages1
Disadvantages2
Spontaneous mutation
no/little regulation
low frequency / restricted choice
Induced mutation
no/little regulation
low frequency / restricted choice
Mutagenic oligonucl.
specific, quick, little/no
regulation
restricted choice
Transposon
may be specific
may be reversible, single target, low
frequency
T-DNA insertion
specific /irreversible
single target, low frequency
Antisense RNA
specific, dominant, sequencebased, many targets
silenced gene intact (reversible), may
be leaky
RNAi (hpRNA)
specific, dominant, sequencebased, many targets
silenced gene intact (reversible), may
be leaky
miRNA
specific, dominant, sequencebased, many targets
silenced gene intact (reversible), may
be leaky
 Each method has pros & cons
 None suites all situations
 Hard or impossible distiguish natural / non-natural
Transcription
Direct gene inactivation
 The gene is mutated (becomes
non functional)
Protein
 RNA missing or aberrant
 Protein missing or non functional
 Usually irreversible
toxin
‘good’ phenotype
‘Classical’ mutation
 Mutations arise spontaneously
in any organism (endogenous or
environment)
 Frequency can be enhanced by
various treatments: UV, X and γrays, chemical mutagens, and
mitogens (indirectly)
Just one base change
out of 15,000
 Crop plants accumulated many
mutations
Konishi et al., (2006) Science 312:1392-1396
Identification of acyanogenic forage Sorghum by a combination of biochemical
screening and TILLING (by Cecilia Blomstedt) ISB News Report, Feb. 2012, p.9-10
One line was totally cyanide deficient
(tcd1) in both shoot and root tissue
throughout all stages of growth and
development. Molecular modeling, based
on the solved crystal structure of relevant
P450s, indicated that the tcd1 mutation, a
proline to leucine amino acid change, is
believed to interfere with the structural
organization of the CYP79A1 protein and
prevent substrate binding and
subsequently a loss of catalytic activity.
…confirmed the complete loss of
CYP79A1 activity and dhurrin production
Sorghum mutant lines growing in the field in Queensland, Australia (~9
weeks old) (A) The M5 generation of selected mutant lines of specific interest,
including the tcd1 and acdc1-3 mutants, growing in the field for seed collection to
be used in future experiments. (B) acdc1 mutant ~0.8m tall. (C) tcd1 mutant
~0.6m tall.
Insertional mutagenesis
Transposons: genetic elements able to jump around in
the genome
Inactivate
genes by:
Retroviruses: virus making new copies (through RNA)
able to integrate into the genome.
T-DNA: bacterial DNA inserted into the plant genome
ATG
T-DNA
STOP
Gene = meaningful sentence
A large bit of DNA ‘breaks’ the gene
 sentence meaning is lost or altered
Gene silencing
petunia expressing a maize
gene
The presence of multiple copies
of the maize gene causes partial
or complete silencing of an
endogenous gene
Antisense RNA
Transcription
Transcription
Duplex formation
Block of
translation
Protein
Indirect gene inactivation
Protein
 The gene is intact but its expression inhibited
toxin
 RNA is either missing, destroyed or non functional
 Another
gene is responsible for the change
‘good’ phenotype
 Only knowledge of the sequence required
toxin
Gossipolo, un esempio illuminante
 Cotton produces 1.65 kg of seed for 1 kg
of fiber
 Due to gossypol, a cardio- and hepatotoxic
terpenoid  seed unfit for consumption by
humans and monogastric animals)
 Seed contains 21% oil and 23% highquality protein
Sunilkumar et al. (2006) P.N.A.S. 103:18054–18059
Cottonseed may help feed the world
 Used as feed for ruminant animals (whole
seeds or meal after oil extraction)
 44 million metric tons (Mt) of cottonseed
(9.4 Mt of protein)
 Could fulfill protein requirements of half a
billion people each year (50 g/day rate)
Sunilkumar et al. (2006) PNAS 103:18054-9
RNAi inactivating δ-cadinene
synthase in the seed
Chlorophyll
Carotenoids
…
Proposed biosynthetic pathway of gossypol
Sunilkumar et al. (2006) PNAS 103:18054-9
RNAi construct for δ-cadinene
synthase
T-DNA region of the vector used to transform cotton
Sunilkumar et al. (2006) PNAS 103:18054-9
Gossypol content of seeds in
individual T1 lines
Levels of gossypol in pooled samples of 30 mature T1 seeds from 26 transgenic lines
Individual transgenic seeds
showed up to a 99% reduction
in compared with wild type
Sunilkumar et al. (2006) PNAS 103:18054–18059
 a strong reduction of gossypol in seed
Levels of gossypol (mg/mg seed) for each individual seed
Sunilkumar et al. (2006) PNAS 103:18054-9
Gossypol (G)
Gossypol and other protective terpenoids are not reduced in leaves,
floral organs, and roots
hemigossypolone (HGQ)
total heliocides (H)
Sunilkumar et al. (2006) PNAS 103:18054–18059
Transgenic seed exhibits a large reduction in Gossypol level.
A monogenic trait: the reduced gossypol trait cosegregates with
the transgene
 Much more predictable, stable, specific
Sunilkumar et al. (2006) PNAS 103:18054–18059
No systemic reduction of gossypol and other protective terpenoids
Spatial and temporal confinement of RNAi-mediated suppression of the gene
Sunilkumar et al. (2006) PNAS 103:18054-9
Ultra-low gossypol cottonseed: generational stability of the
seed-specific, RNAi-mediated phenotype and resumption of
terpenoid profile following seed germination
Rathore (2012) Plant Biotechnology Journal 10:174-183.
…unlike the unstable nature of antisense-mediated low seedgossypol phenotype, the RNAi-mediated ULGCS trait exhibited
multi-generational stability.
Transgenic vs. conventional
 A glandless mutant was obtained with conventional strategies.
Varieties with this trait were a failure under field conditions
(extraordinarily susceptible to a host of insect pests)
 Terpenoids protect the plant from both insects and pathogens
 The transgenic approach achieved a goal classical breeding was
unable to obtain (specific reduction in seed)
Targeted gene silencing can be used to modulate biosynthetic
pathways in a specific tissue to obtain a desired phenotype.
Impossible by traditional breeding
 Texas A&M University and U.S. Department of Agriculture
Sunilkumar et al. (2006) PNAS 103:18054–18059
Take home message (I)
“[this] approach…not only improves food safety but also
provides an additional and potentially extraordinary mean
to meet the nutritional requirements of the growing world
population without having to increase either crop yields
or acreage planted”
(Sunilkumar et al., 2006)
“Our hope is to get through regulatory approval process in the U.S. first.
However, it takes $50-100 million to go through the process. At this
point, we don't know where the money is going to come from, but we are
exploring various possibilities. Getting U.S. approval will make it easier
to then get permit in other countries. We will be especially interested in
some African countries and some Asian countries. “
(personal communication by Keerti S. Rathore)
Gene technology could improve food safety, food security and
reduce environmental impact. Regulation is a major obstacle
Lathyrus
sativus
A hardy tropical/subtropical legume
Important source of nutrition but contains
a neurotoxin: oxalyldiamino-propionic
acid (ODAP)
http://www.treknature.com/gallery/Asia/India/photo152618.htm
http://www.gudjons.com/Mittel/Lathyrus-tub.jpg
http://www.grainlegumes.com/fckeditor/aepfiles/File/Species/Lathyrus_sativus_pod_(L.delaRosa)_600.jpg
 Beans from this so-called “famine crop” (consumed by poor people in Asia and
Africa) causes lathyrism.
 a paralytic disease (spastic paraparesis) prevalent among adults in Central
India who have consumed large quantities of L. sativus seeds for several months
 Safe content for ODAP is < 0.2%. Content in varieties range: 0.30-3.3
 Classical breeding approaches are in progress, but what about a transgenic
approach targeting the toxin biosynthetic pathway only in the seed?
Biosynthesis
Proposed biosynthetic pathway for β-1 in Lathyrus sativus
(products in brackets have not been detected)
Yan et al., (2006) Phytochemistry 67:107–121
Fonio & pearl millet cause goiter
Gressel (2008) Genetic Glass Ceilings
Cyanide in Cassava
 4th most important source of calories in the tropics.
 Staple for about 800 million people worldwide.
 Average annual per capita consumption of cassava
in 2003 was 300 kg in Dem. Rep. Congo.
 Processing (and cooking) to reduce cyanide levels,
but results in loss of proteins, vitamins, and minerals.
 Cyanide-associated health disorders have been
attributed to eating poorly processed cassava
http://www.food-info.net/uk/products/rt/cassava.htm
How to reduce cyanide content?
http://thekebun.wordpress.com/2008/
10/01/feeding-your-goats-cassava/
Siritunga et al., (2004) Plant Mol Biol. 56:661-9
A strong reduction in root but not in leaf would be desirable
http://www.fao.org/Wairdocs/ILRI/x5458E/x5458e0b.htm
Transgenic plants show a
strong reduction in leaf and
root cyanogenic glucoside
(linamarin) content
Transformants with large reductions
(94% -60%) in leaf linamarin content
all had root linamarin contents that
were less than 1% of wild-type.
BUT  plants impaired in growth or tuber
formation presumably due to the role of
cyanogen hydrolysis in aminoacid
biosynthesis.
A more promising strategy: expressing the
leaf-specific enzyme hydroxynitrile lyase
(HNL) in roots in order to accelerate
cyanogenesis and cyanide volatilization
during processing.
Another is to increase protein accumulation
Siritunga et al., (2004) Plant Mol Biol. 56:661-9.
controlli
Esprimendo proteine nelle radici si
migliora la qualità nutrizionale e si
riducono i glucosidi cianogenici
Transgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta)
Generates a Novel Sink for Protein. Abhary (2011) PLoS ONE 6:e16256.
Mycotoxins
Courtesy of T. Maggiore
 Mycotoxins in grains are a major health
problem (fumonisins and aflatoxins)
 Cause cancer and neural tube defects
 Improve resistance or increase degradation
- direct: improve resistance to fungi (corn expressing plant defensin in field
trials with encouraging results)
- Engineer mycotoxin degrading activities
- indirect: reduce insect damage through Bt toxin (effective for fumonisins)
Courtesy of K. Petroni
Neural tube defects:
Epidemiological data
Data courtesy of Dr. Julio Cabrera, 2008
Implicate fumonisins in maize as one of the causal
factors
- Average Guatemalan eats 115 kg-1 ∙ year
- FB1 Fumonisins average 1.2 ppm
- Can be as high as 6 ppm
- FB1 Calculated Daily Intake
- 7.2 μg kg-1bw d-1(average woman)
Slide courtesy of Dr. Wayne Parrott
Slide courtesy of Dr. Wayne Parrott
Slide courtesy of Dr. Wayne Parrott
Courtesy of K. Petroni
Tonelli, Pilu, Petroni
(University of Milan, IT)
Classical breeding
(Flavonoid and
anthocyanins)
Some colored lines show lower levels of fumonisin
compared to control yellow lines
Transgenesis
(Bt maize)
Other examples by classical breeding
Erucic acid in Brassica napus
Cyanogenic glucosides in trifolium
Glucosinolates in brassica
Many food security/safety problems in the developing
world are waiting for a solution. Genetic manipulation is
more precise, more predictable, low cost solutions
(not necessarily alternative to breeding)
Consequences of toxin reduction
Reducing glucosinolates in Arabidopsis
Glucosinolates are sulphur rich
compounds from brassicas
Some beneficial, other toxic (quantity!)
Upon wounding are converted into toxic
products
Regulators identified (two branches)
Mutants isolated
Short chain
Aliphatic GSL
Long chain
Indolic GSL
Beekwilder et al., (2008) PLoS 3:e2068.
Mutating Myb28 and Myb29
Regulators
Beekwilder et al., (2008) PLoS 3:e2068.
Reducing glucosinolate content...
...stimulates pest growth and damage!
Beekwilder et al., (2008) PLoS 3:e2068
A. thaliana making
cyanogenic glucosides
A) Adult beetles fed extensively only on leaves
Effect onprocess:
flea beetle
Reduction
in
toxin
content
is
a
trade-off
containing no dhurrin. B) Larvae frequently initiated
and
larvae feeding
susceptibility
to pests
no mines on leaves increases
containing dhurrin,
although
attempts were made to feed (indicated by circles) Tattersall et al., (2001) Science 293:1826-8
Effect on larvae
Nearly all larvae (98%) presented
to leaves containing about 4 mg
of dhurrin/gfw died.
Transgenic A. thaliana plants
released high levels of HCN, up to
2 μmol/gfw, upon tissue damage.
An endogenous β-glucosidase
with dhurrin hydrolyzing activity
is present in A. thaliana.
Consumption of leaf-disc material from the transgenic lines expressing the two cytochrome P450
genes (CYP79A1 and CYP71E1), or the UDPG-glucosyltransferase gene (sbHMNGT), or containing
the two empty expression vectors was not significantly different from the consumption of leaf-disc
material from wt plants.
Tattersall et al., (2001) Science 293:1826-8
Life is full of trade-offs
Reduction in pesticide content (a corollary of
crop domestication) is not without
consequences!
 General pesticides (e.g. cyanide) are worse
for humans than specific ones (es. Bt)
Eat the pesticides you prefer (natural does
not imply safer)
Take home message (II)
Allergens
 Widespread occurrence
 You may not know it until you experience it
 Nuisance / cost / deadly threat
 Minute amounts of allergens may cause a lifethreatening anaphylactic reaction
 May occur after ingestion, skin contact, injection
of an allergen or inhalation.
 48 deaths caused by food over a 7-year period
between 1999 and 2006 in UK
Anaphylactic shock: when food kills www.youtube.com/watch?v=XC0nHFblLcE
Allergies caused by plants
Eight foods account for 90% of all food-allergic reactions.
milk, egg, fish, shellfish, peanut, tree nut, soy, wheat
Pollen is the major cause of respiratory allergy.
At least 40% of type 1 allergic patients are sensitized
against grass pollen allergens
 Contrary to common perception, transgenic plants never
caused allergic reactions to consumers. Many conventional
crops do it regularly
 If a gene used for transgenesis comes from a plant
containing allergens, the transgene is checked for allergenicity
Transgenesis, rather than a cause of allergy, can be part of the solution
Reducing plant allergens






Apple
Peanut
Wheat (celiac disease)
Soybean
Ryegrass
Birch
Soybean allergen: P34
US/Europe: 5 - 8% of babies and 2% of adults allergic* to soybeans
Dominant soybean allergens is P34: > 65% of soy-sensitive patients react only
to P34 protein
Transgenic soybean without P34 published in 2003 (Herman et al., 2003)
Herman et al., (2003)
Plant Physiol. 132:36-43
No difference in composition, development, structure, or ultrastructure when
compared with control plants. No other significant changes in polypeptide pattern
“Regulatory difficulties and the lack of acceptance of GM soybeans by the baby
food and formula industry makes using such an allergen-suppressed soybean
difficult [read impossible] at the present time.”
Alternative approach: identify soybeans with little/no allergen
 screen the entire USDA national soybean germplasm collection
Joseph et al. (2006) 46:1755-63
Out of > 16 266 accessions soybean germplasm screened,
12 lines (2 in the cultivated soybean) have no P34 allergen
Why these two soybean plants lack the antigen?
Apple major allergen: Mal d1
Apple allergy is dominated by protein Mal d 1 where birch pollen is endemic
 RNAi Mal d 1 in apple plants transgenically
expression successfully reduced in vivo allergenicity
Gilissen et al. (2005) J. Allergy Clin. Immunol. 115:364-369
Allergenicity depends on the presence
and amount of some specific Mal d 1
isoforms.
Classical breeding allows creation
new hypo-allergenic cultivars
Gao et al. (2008) BMC Plant Biology 8:116
Where logic ends, biotech regulation begins
If a protein is > 50% identical to an allergenic protein, it is a
“potentially allergen”.
Transgenic products have to be labelled
Phaseolin
► eaten by one billion people everyday
► NOT recognized as an allergen
BUT
► 54% similar to conglycinin (a minor soybean allergen)
► ‘potential allergen’ according to biosafety regulation
A transgenic plant expressing Zeolin phaseolin or a fusion thereof
would require labelling (often impossible and/or ridiculous in many
countries) because of phaseolin similarity to a know allergen is 54%
Two texts with 60% similarity
The glory of Him who moveth everything
Doth penetrate the universe, and shine
In one part more and in another less
(Dante, Paradise,
I, v.1-3)the same effect
60 % similarity
does Canto
not give
The story of him who believeth everything
Does perpetuate diverse lies and causes
one part of farmers or another to die
New almond or peach varieties may accumulate much more cyanogenic
glucosides, new potato varieties may accumulate more or new glycoalkaloids.
In Italy and the EU they require no regulatory scrutiny (no compulsory tests)
before release, cultivation or commercialization if they are produced by
conventional breeding or mutagenesis.
Conclusions
Plant derived allergens and toxins are
ubiquitous, abundant
Tools are available to reduce them
(conventional or transgenic)
Strategies must be reasonable (accept
some level of risk) [risks / benefits]
Overcautious regulation kills the
technology and associated benefits
Time to say the truth
A man came home at noon, said good night to everybody, and went to
bed. His wife, much concerned, asked him if he were ill, and the good
man answered: 'I am quite well. But this morning everybody told me
that I was drunk. I am not drunk -- you know that I do not even take
wine, that I am an abstainer -- but I love peace, and in order not to
contradict them I am going to bed.'
Plant biotechnologists avoided discussions (complied to insane
regulations) for the sake of peace.
Time has come to raise the voice and demand a change
Present regulation is unscientific, very costly, excessive, restricting the
potential uses of the technology to few crops and is aborting the technology
in and for developing countries.
Propagation of unreasonable and unfounded fears about biotechnology
mantains a high regulatory burden and keeps de facto the technology in the
hand of few industrial groups which are interested only in few crops/problems
The P38 and the apple
A true desire, when does not come to terms with reality, but follows the path of
irrational utopia, turns into lie and can only end up in murderous madness and
self destruction.
S. Allevato e P. Cerocchi (2009) “La P38 e la mela”, publ. ITACA, p.173
Un desiderio vero, quando non fa i conti con la realtà,
ma imbocca la strada dell’utopia irrazionale, diventa
menzogna, e non può che condurre alla follia omicida e
all’autodistruzione.
S. Allevato e P. Cerocchi (2009) “La P38 e la
mela”, ed. ITACA, p.173
Acknowledgements
• Support or material from colleagues and
friends (especially Parrot, Gressel
Kershen, Salamini, Fico, Vitale, Ederle,
Maggiore, Petroni, Rossi…)
• Confidential information or help with
literature (Gilissen, Bisht, Rathore, Shah,
Carputo, Parisi, Faquet …)
• The support of the CSBA (access to
specific books) and Univ. of Milan
Bibliography silencing/mutations
• Carlini and Grossi-de-Sa´ (2002) Toxicon 40:1515–1539
• R Koes et al. (1995) Targeted gene inactivation in petunia by PCRbased selection of transposon insertion mutants. Proc Natl Acad Sci
U S A. 92:8149-8153.
• Hamilton AJ, Baulcombe DC (1999) A species of small antisense
RNA in posttranscriptional gene silencing in plants. Science 286:
950–952
• Batista
• Napoli et al. (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
• van der Kroll et al. (1988) An anti-sense chalcone synthase gene in
transgenic plants inhibits flower pigmentation Nature 333:866-869
References classical mutants
* Li et al. (2006) Rice domestication by reducing shattering. Science
311:1936-1939.
* Konishi et al. (2006) An SNP caused loss of seed shattering during rice
domestication. Science 312:1392-1396.
* Simons et al. (2006) Molecular characterization of the major wheat
domestication gene Q. Genetics 172:547-555.
* Wright et al. (2005) The effects of artificial selection of the maize
genome. Science 308, 1310–1314.
* Peng et al., (1999) 'Green revolution' genes encode mutant gibberellin
response modulators. Nature 400:256-61.
* Hedden P. (2003) The genes of the Green Revolution. Trends Genet.
19:5-9.
Dangers breeding/natural
 Lenape: Name of potato variety Lenape withdrawn . Am. J. Potato Res. (1970) 47:103
http://www.springerlink.com/content/a6k677974316541m/fulltext.pdf?page=1
 Herrington (1983) Intense Bitterness in Commercial Zucchini. Cucurbit Genetics
Cooperative Report 6:75-76 http://cuke.hort.ncsu.edu/cgc/cgc06/cgc6-38.html
 Sharma (2006) Bottle Gourd Poisoning. Journal of Medical Education & Research, 8:120121. ISSN 0972-1177. http://openmed.nic.in/1710/01/r.letter.pdf
 Browning S, Hodges L. Cucumber crop information: Bitterness in Zucchini Squash and
cucumber. http://cuke.hort.ncsu.edu/cucurbit/cuke/cukehndbk/cukebitterness.html
 Finkelstein et al., (1994) An outbreak of phytophotodermatitis due to celery. Int. J.
Dermatol.33:116-8.
 Fleming (1990) Dermatitis in grocery workers associated with high natural concentrations
of furanocoumarins in celery. Allergy Proc. 11:125-7.
 Berkley et al., (1986) Dermatitis in grocery workers associated with high natural
concentrations of furanocoumarins in celery. Ann Intern Med. 105:351-5.
 Ames et al., (1990) Dietary pesticides (99.99% all natural). Proc. Nad. Acad. Sci. USA
87:7777-7781.
 Ames B.N., Profet M., and Gold L. S. (1990) Nature's chemicals and synthetic chemicals:
comparative toxicology. PNAS 87:19 7782-7786.
Hansen A. A. (1925) Two fatal cases of potato poisoning Science 61:340-341
Sengul et al., Food Control 15, 281-286. Storage conditions of potato on glycoalkaloids. lots put it up
particularly wounding
Wilson, GS. Mon. Bull.Minist.Health Laboratory service. 18, 207-210.(1959) A small outbreak of
solanine poisoning. References other cases too.
Lee et al., J Agric Food Chem. 52, 2832-2839 (2004). Glycoalkaloids and metabolites inhibit the growth
of human colon and liver cancer cells.
Korpan et al., Trends in Biotech. 22, 147-151. (2004)Glycoalkaloids. True Safety or false sense of
security.
Smith et al., Trends in Food Science and Technology. 7, 126-131. (1996) Potato glycoalkaloids:some
unanswered questions.
Willimott. The Analyst. LVIII no 689. 431-439.(1933) An investigation of solanine poisoning.
This paper refers to cases in 1917. 1902. 1928. 1899. 1932
Claringbold et al., Xenobiotica 12, 293-302. (1982) kinetics and retention of solanidine in man. Lasts
years after one meal. If DDT retention is of concern so is this.
Morris and Lee. Food Technology in Australia. 36, 118-124. (1984) Total toxicity and teratogenicity of
solanaceal glycoalkoids.
Friedman and McDonald. Crit Revs in Plant Science 16, 55-132. (1997). Glycoalkaloids :chemistry
analysis , safety and plant physiology.
McMillan and Thompson. Quarterly Journal of Medicine. XLVIII no 190. 227-243. (1979). An outbreak of
suspected solanine poisoning in schoolboys.
Info from W. Parrott (March 2013)
Actually, the Lenape withdrawal notice says its bitter taste served as notice that something was amiss, and this was still during
the seed increase phase in preparation for commercial release.
Overall, the tale of the Lenape potato is a terrible and misleading example to use as an example of the dangerous unintended
consequences that genetic modification can have.
In first place, glycoalkaloids are a totally known hazard in potatoes. Secondly, Lenape was derived from a cross to a potato
species known for its high alkaloid levels.
Thirdly, it is now customary for all known toxins and antinutrients to be monitored during breeding and transgenic programs.
Such known toxins are routinely listed in the OECD list of metabolites that are tested for transgenics, as specified by Codex.
The current fear about unintended/unknown hazards with transgenics has nothing to do with known hazards such as
glycoalkaloids as in the Lenape example. The problem comes from the hypothetical presence of unknown toxins, that would
originate from the awakening of long-dormant pathways brought back to life by the stress or mutations of the transformation
process.
Obviously, there is no test for the presence of something totally unknown and which could be from any number of chemical
categories. Thus, one aim of the "substantial equivalence" paradigm is to infer the presence of such toxins through changes in
composition (indicating a metabolic shunt elsewhere). Whole food studies are also done for this
purpose. Thus, there is a multimillion dollar expense added to the cost of transgenic safety review.
There was a particularly interesting presentation at last fall's crop composition workshop sponsored by ILSI-IFBiC: see
http://www.youtube.com/watch?v=AyeS96yda3I&amp;feature=youtu.be
The bottom line: of some 70 dossiers submitted to FDA, none showed any relevant differences in composition.
In addition, another of the ILSI-IFBiC task forces did a literature review, which just got published in Plant Physiology:
http://www.plantphysiol.org/content/early/2013/03/04/pp.112.209817.full.pdf+html?sid=caf1ee16-ebad-4614-adb66a318ead3ad6
The bottom line, in all the history of breeding, mutation breeding, wide hybrids, etc., all cases of crop toxicity have involved
*known* toxins. There is not a single example of a previously unknown toxin for the genus arising spontaneously. Thus, I do not
see what is so different about genetic engineering when it comes to reviving toxin metabolic pathways.
So the idea that millions must be spent on GM safety testing for unintended, unexpected consequences from *unknown*
hazards is baloney. We will always continue to test for known hazards, regardless if the crop is biotech or conventional.
As a comment on http://boingboing.net/2013/03/25/the-case-of-the-poison-potato.html
Phytate
•
•
•
Campion et al., (2009) Isolation and characterisation of an lpa (low phytic
acid) mutant in common bean (Phaseolus vulgaris L.). Theor Appl Genet.
2009 Feb 18.
Shi et al. (2007) Embryo-specific silencing of a transporter reduces phytic
acid content of maize and soybean seeds Nature Biotechnology 25, 930937
Doria et al. (2009) Phytic acid prevents oxidative stress in seeds: evidence
from a maize (Zea mays L.) low phytic acid mutant. J Exp Bot. 60:967-78.
Phytate has a negative impact on animal nutrition and the environment. Shi et al. produced soy and
maize with a reduced phytate content
Gene inactivation
* Sunilkumar et al. (2006) Engineering cottonseed for use in human nutrition
by tissue-specific reduction of toxic gossypol. PNAS 103:18054-9
* Siritunga and Sayre (2003) Generation of cyanogen-free transgenic cassava
Planta 217: 367–373.
* Beekwilder et al. (2008) The impact of the absence of aliphatic
glucosinolates on insect herbivory in Arabidopsis. PLoS ONE. 3:e2068.
Other examples:
* Lewis et al., (2008) RNA interference (RNAi)-induced suppression of
nicotine demethylase activity reduces levels of a key carcinogen in cured
tobacco leaves. Plant Biotechnol J. 2008 Feb 14;
Lathyrus & Millet
Yan et al., (2006) Lathyrus sativus (grass pea) and its neurotoxin ODAP.
Phytochemistry 67:107–121 (review)
“Swollen Necks from Fonio Millet and Pearl Millet”, chapter 18 of Genetic
Glass Ceilings (J. Gressel) 2008
 Allen et al., (2004) RNAi-mediated replacement of morphine with the
nonnarcotic alkaloid reticuline in opium poppy. Nat Biotechnol. 22:1559-66.
 Liu Q, Singh SP, Green AG. (2002) High-stearic and High-oleic cottonseed
oils produced by hairpin RNA-mediated post-transcriptional gene silencing.
Plant Physiol. 129:1732-43.
 Stoutjesdijk PA et al., hpRNA-mediated targeting of the Arabidopsis FAD2
gene gives highly efficient and stable silencing. Plant Physiol. 129:1723-31.
 Grubb CD, Abel S. (2006) Glucosinolate metabolism and its control. Trends
Plant Sci. 11:89-100.
 Stevenson-Paulik et al., (2005) Generation of phytate-free seeds in
Arabidopsis through disruption of inositol polyphosphate kinases. Proc Natl
Acad Sci U S A. 102:12612-7.
 Tattersall DB et al., (2001) Resistance to an herbivore through engineered
cyanogenic glucoside synthesis. Science 293:1826-8.
Allergens
•
E. N. Clare Mills (Editor), Peter R. Shewry (Editor) "Plant Food Allergens“ (2005)
Wiley-Blackwell. ISBN-10: 0632059826
•
Gilissen et al., (2005) Silencing the major apple allergen Mal d 1 by using the RNA
interference approachJ. Allergy Clin. Immunol. 115:364-369
Gao et al. (2008) Assessment of allelic diversity in intron-containing Mal d 1 genes
and their association to apple allergenicity. BMC Plant Biology 8:116
Herman et al., (2003) Targeted gene silencing removes an immunodominant allergen
from soybean seeds. Plant Physiol. 132:36-43
Herman E. (2005) Soybean Allergenicity and Suppression of the Immunodominant
Allergen. Crop Sci. 45:462–467
Schenk M. (2008) Birch pollen allergy: molecular characterization and hypoallergenic
products. Ph.D. thesis (Wageningen University). ISBN 978-90-8504-873-2
Pumphrey RS, Gowland MH. Further fatal allergic reactions to food in the United
Kingdom, 1999-2006. J Allergy Clin Immunol 2007;119:1018-9.
Anaphylactic shock: http://www.youtube.com/watch?v=XC0nHFblLcE
Yaklich, R., R. Helm, and E. Herman. 1999. Analysis of the distribution of the major
soybean allergen in a core collection of Glycine max accessions. Crop Sci. 39:1444–
1447.
Joseph L.M., Hymowitz T., Schmidt M.A. and Herman E. M. (2006) Evaluation of
Glycine Germplasm for Nulls of the Immunodominant Allergen P34/Gly m Bd 30k.
Crop Sci 46:1755-1763
•
•
•
•
•
•
•
•
Ref. toxicity
Cassava poisoning
http://www.aciar.gov.au/project/CS2/1990/007
http://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_19_01.html
Teles FF. (2002) Chronic poisoning by hydrogen cyanide in cassava and its prevention in
Africa and Latin America. Food Nutr Bull. 23:407-12.
Oluwole et al., (2000) Persistence of tropical ataxic neuropathy in a Nigerian community. J.
Neurol. Neurosurg. Psychiatry 69:96-101.
http://www.foodstandards.gov.au/_srcfiles/28_Cyanogenic_glycosides.pdf
(glucosidi cianogenici)
http://www.ripper.com.au/~enneking/pdf/pdf-Famine-in-Afghanistan-threat-of-a-newlathyrism-epidemic.pdf (Lathyrism)
http://encyclopedia.farlex.com/cassava (Cassava)
Useful sites
•
www.botgard.ucla.edu/html/botanytextbooks/economicbotany/index.html
•
•
Poisoning
http://www.biomedexperts.com/Abstract.bme/17560071/Acute_Datura_stramonium_poisoning_in
_an_emergency_department
•
http://www.uni-sz.bg/tsj/Vo4No3_1/Binev%202.pdf
When too much becomes little
RNA silencing of GUS in rice callus using a sense, antisense, or IR vector.
Waterhouse et al. (1998)
The presence of a dsRNA is the crucial element
Eamens (2008) Plant Physiol.147:456–468
Gene to be silenced
RNA-mediated
Once you know the sequence of a
gene, it becomes easy to inactivate
- only that gene
- only in certain tissue/organ
Mansoor et al., (2006) Trends Plant Sci. 11:559-65.
RNAi- mediated pathways in plants (2005)
Si conoscono
molti geni
implicati nel
silenziamento