Una visione sistematica per l’ingegneria metabolica
Cosa ha da dire la MCA ai biotecnologi vegetali?
Ingegneria metabolica probabilmente efficace probab. inefficace
FQ
E
(1)
P
C
A
B
(2)
A
B
(3)
P
C
A
B
(4)
P
C
A
B
(5)
P
C
A
B
(7)
P
C
A
B
Q
TF+
SS S S S S
(8)
P
C
A
B
S
+
(6)
P
C
A
B
S
PATP
Z
W
Y
X
(9)
P
C
A
B
S
+
+
Flux change: an asymmetric problem
Inattivazione di enzimi
Spegnimento, parziale o totale, di geni tramite:
- Knock out
- Antisenso
-Cosoppressione
-RNA interference
(2)
A
B
S Glucosinolati (Cyt. P450)
Biosintesi di lignina
Ac. grassi poli-insaturi (desaturasi)
Morfina (COR)
Caffeina
Lisina
Amido
......
Domesticazione?
Inactivation of Allergens and Toxins
Piero Morandini
Dept. of Biology
Milan University (Italy)
Per approfondire: manoscritto con 150 referenze
Outline
The dangers of nature and foodGene inactivation strategiesManipulating crops (toxins)Manipulating crops (allergens)Transgenic vs. classical approachesConsequences & 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)
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
precise map metal detector
Plant Toxic substance Effect DoseAssa 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 20 seeds
Brassicas Glucosinolates toxic
A field trip into the ‘mine field’
Focus mainly on small metabolites (but toxic proteins also abound in nature)
Solanum tuberosum
http://commons.wikimedia.org/wiki/File:Potato_sprouts.jpg
Poisoning and Toxicology Handbookby Leikin & Paloucek 4th edition, Informa Health Care, 2007ISBN 1420044796, 9781420044799
Solanine abounds in green parts, sprouts and diseased potatos
March 27, 1925 340-341
http://depthofprocessing.blogspot.com/2009/05/are-potato-peels-nutritious.html
http://kanaya.naist.jp/knapsack_jsp/image.jsp?word=C00002265
Solasonine
Solasonine accumulates in Solanum sodomeum
LD50=30 mg/kg
Datura stramoniumangel'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 annuumpeperoncino
* 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
www.floralimages.co.uk/pdaturstram1.htm
http://www.ubcbotanicalgarden.org/potd/2006/10/capsicum_annuum_cultivars.php
http://www.luciolongo.it/Fotografie%20strane%20che%20ho%20fatto%20in%20giro.htm
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 16-year-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)…
Case report: http://www.cjem-online.ca/v9/n6/p467
Teenagers with Jimson weed (Datura stramonium) poisoning
http://uvalde.tamu.edu/herbarium/final/dain_fr.jpg
* 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.
http://en.wikipedia.org/wiki/Psoralen
Celery contains several natural pesticidesXanthotoxin or 8-methoxypsoralen
Psoralens form adducts with DNA upon exposure to light and therefore cause photodermatitis (= light dependent dermatitis)
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.
Giant hogweed
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://en.wikipedia.org/wiki/File:Colchicum_speciosum000.jpg
http://en.wikipedia.org/wiki/Colchicine
http://pandasthumb.org/archives/2010/08/08/Samarasinghe.Gloriosa_superba.JPG
Colchicine is a potent poison: it blocks microtubule formation and thereore separation of chromosomes at cell division.
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 43:399Poisoning 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.
Latin name Toxic substance1 Effect2 Dose3 | content4
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 | 3-6%
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 | 0-2 mg/g
Solanum lycopersicum Tomatine toxic
Solanum tuberosum Solanine lethal | 3-6 mg/Kg
Manihot esculenta Cyanogenic glucosides paralysis-stunting | 15-400 mg HCN/kg
Glycine max Protease / amylase inhibitors toxic
Prunus dulcis Cyanogenic glucosides lethal 20 seeds | 29 mg/kg
Brassica oleracea Glucosinolates lethal-goiter | 1-2 mg/g
Gossypium hirsutum Gossypol cardio/ hepatotoxic 0.3-3 mg/kg | 10 mg/g
Lathyrus sativus oxalyl-diaminopropionic acid neurotoxin /paresis | 0.3-3.2%
Phaseolus lunatus Cyanogenic glucosides lethal | 2-3 mg HCN /kg
Papaver somniferum Morphine lethal 100 mg | 10 mg/g
Several species Cyanogenic glucoside toxic | 1-8 g HCN /kg
Mycotoxins
Maize, peanut Aflatoxins Cancer 1-5 mg/kg
Maize Fumonisins Cancer / spina bifida
A matter of dose (threshold) reduction below a threshold considered safe
The issue of toxic substances in plants is not new nor is gone
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)…
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)
http://www.springerlink.com/content/37827612x62xp348/
Bottle gourd Celery
http://www.people.cornell.edu/pages/kjc34/distribution.html
http://nonsense123.files.wordpress.com/2008/06/bottle-gourd.jpg
Zucchini Blackjack
http://www.growfruitandveg.co.uk/grapevine/vegging-out/courgettes-doing-my-head_18095.html
Which approaches?
How to reduce toxic substances in plant? Target the gene(s)
Gene RNA Protein
Toxins are either proteins or are produced through proteins
Cucurbitacins in Plant Food Di Jørn Gry, Inge Søborg and Hans Christer Andersson
Recupera formule di struttura...
Libro su goggle books
Gene inactivation strategies
Regulator
a) inactivate a protein (enzyme)
Halkier (2006) Annu. Rev. Plant Biol. 57:303-33
Degradation product
b) increase toxin degradation
c) target a regulator of the synthesis
Tools of the tradeto 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, sequence-based, many targets
silenced gene intact (reversible), may be leaky
RNAi (hpRNA) specific, dominant, sequence-based, many targets
silenced gene intact (reversible), may be leaky
miRNA specific, dominant, sequence-based, 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
Protein
toxin
Transcription
‘good’ phenotype
Direct gene inactivation
The gene is mutated (becomes non functional)
RNA missing or aberrant
Protein missing or non functional
Usually irreversible
‘Classical’ mutation Mutations arise spontaneously in any organism (endogenous or environment)
Frequency can be enhanced by various treatments: UV, X and γ-rays, chemical mutagens, and mitogens (indirectly)
Crop plants accumulated many mutations
Just one base change out of 15,000
Konishi et al., (2006) Science 312:1392-1396
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.
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
Identification of acyanogenic forage Sorghum by a combination of biochemical screening and TILLING (by Cecilia Blomstedt) ISB News Report, Feb. 2012, p.9-10
Insertional mutagenesisTransposons: genetic elements able to jump around in the genome
Retroviruses: virus making new copies (through RNA) able to integrate into the genome.
T-DNA: bacterial DNA inserted into the plant genome
A large bit of DNA ‘breaks’ the gene
sentence meaning is lost or altered
Inactivate genes by:
STOP
Gene = meaningful sentence
ATGT-DNA
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
Protein
toxin
Transcription
Duplex formation
Block of translationProtein
toxin
‘good’ phenotype
Indirect gene inactivation
The gene is intact but its expression inhibited
RNA is either missing, destroyed or non functional
Another gene is responsible for the change
Only knowledge of the sequence required
Transcription
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% high-quality 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
Proposed biosynthetic pathway of gossypol Sunilkumar et al. (2006) PNAS 103:18054-9
Chlorophyll
Carotenoids
…
RNAi inactivating δ-cadinene synthase in the seed
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
Sunilkumar et al. (2006) PNAS 103:18054–18059
Individual transgenic seeds showed up to a 99% reduction in compared with wild type
Levels of gossypol (mg/mg seed) for each individual seed
a strong reduction of gossypol in seed
Sunilkumar et al. (2006) PNAS 103:18054-9
Gossypol and other protective terpenoids are not reduced in leaves, floral organs, and roots
Sunilkumar et al. (2006) PNAS 103:18054–18059total heliocides (H)
Gossypol (G)
hemigossypolone (HGQ)
Transgenic seed exhibits a large reduction in Gossypol level.
Sunilkumar et al. (2006) PNAS 103:18054–18059
A monogenic trait: the reduced gossypol trait cosegregates with the transgene Much more predictable, stable, specific
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
Rathore (2012) Plant Biotechnology Journal 10:174-183.
…unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.
Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination
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)
Sunilkumar et al. (2006) PNAS 103:18054–18059
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
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
http://www.treknature.com/gallery/Asia/India/photo152618.htm
Important source of nutrition but contains a neurotoxin: oxalyldiamino-propionic acid (ODAP)
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?
http://www.grainlegumes.com/fckeditor/aepfiles/File/Species/Lathyrus_sativus_pod_(L.delaRosa)_600.jpg
http://www.gudjons.com/Mittel/Lathyrus-tub.jpg
Proposed biosynthetic pathway for β-1 in Lathyrus sativus (products in brackets have not been detected)
Yan et al., (2006) Phytochemistry 67:107–121
Biosynthesis
Fonio & pearl millet cause goiter
Gressel (2008) Genetic Glass Ceilings
Cyanide in Cassava
http://www.food-info.net/uk/products/rt/cassava.htm
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
How to reduce cyanide content?
Siritunga et al., (2004) Plant Mol Biol. 56:661-9
http://www.fao.org/Wairdocs/ILRI/x5458E/x5458e0b.htm
http://thekebun.wordpress.com/2008/10/01/feeding-your-goats-cassava/
A strong reduction in root but not in leaf would be desirable
Transgenic plants show a strong reduction in leaf and root cyanogenic glucoside (linamarin) content
Siritunga et al., (2004) Plant Mol Biol. 56:661-9.
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
Transgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta) Generates a Novel Sink for Protein. Abhary (2011) PLoS ONE 6:e16256.
Esprimendo proteine nelle radici si migliora la qualità nutrizionale e si riducono i glucosidi cianogenici
con
tro
lli
Mycotoxins Mycotoxins in grains are a major health problem (fumonisins and aflatoxins)
Cause cancer and neural tube defects
- 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)
Improve resistance or increase degradation
Courtesy of T. Maggiore
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
Some colored lines show lower levels of fumonisin compared to control yellow lines
Tonelli, Pilu, Petroni (University of Milan, IT)
Classical breeding
(Flavonoid and anthocyanins)
Transgenesis
(Bt maize)
Courtesy of K. Petroni
Other examples by classical breedingErucic 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
productsRegulators identified (two branches)Mutants isolated
Aliphatic GSL
Indolic GSL
Short chain
Long chain
Beekwilder et al., (2008) PLoS 3:e2068.
Mutating Myb28 and Myb29
Beekwilder et al., (2008) PLoS 3:e2068.
Regulators
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 containing no dhurrin. B) Larvae frequently initiated no mines on leaves containing dhurrin, although attempts were made to feed (indicated by circles)
Effect on flea beetle and larvae feeding
Tattersall et al., (2001) Science 293:1826-8
Reduction in toxin content is a trade-off process: increases susceptibility to pests
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 life-
threatening 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 plantsEight foods account for 90% of all food-allergic reactions. milk, egg, fish, shellfish, peanut, tree nut, soy, wheat
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
Pollen is the major cause of respiratory allergy. At least 40% of type 1 allergic patients are sensitized against grass pollen allergens
Reducing plant allergens
Apple Peanut Wheat (celiac disease) Soybean Ryegrass Birch
Transgenesis, rather than a cause of allergy, can be part of the solution
Soybean allergen: P34US/Europe: 5 - 8% of babies and 2% of adults allergic* to soybeansDominant soybean allergens is P34: > 65% of soy-sensitive patients react only to P34 proteinTransgenic soybean without P34 published in 2003 (Herman et al., 2003)
No difference in composition, development, structure, or ultrastructure when compared with control plants. No other significant changes in polypeptide pattern
Herman et al., (2003) Plant Physiol. 132:36-43
Alternative approach: identify soybeans with little/no allergen screen the entire USDA national soybean germplasm collection
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?
“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.”
Joseph et al. (2006) 46:1755-63
Apple major allergen: Mal d1Apple allergy is dominated by protein Mal d 1 where birch pollen is endemic RNAi Mal d 1 in apple plants transgenicallyexpression successfully reduced in vivo allergenicity
Allergenicity depends on the presence and amount of some specific Mal d 1 isoforms.
Classical breeding allows creation new hypo-allergenic cultivars
Gilissen et al. (2005) J. Allergy Clin. Immunol. 115:364-369
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%
The glory of Him who moveth everythingDoth penetrate the universe, and shineIn one part more and in another less
(Dante, Paradise, Canto I, v.1-3)
The story of him who believeth everythingDoes perpetuate diverse lies and causesone part of farmers or another to die
60 % similarity does not give the same effect
Two texts with 60% similarity
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 truthA 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
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
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
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 PCR-
based 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, 8:120-
121. 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&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-adb6-6a318ead3ad6
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
• 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, 930-937
• 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
Phytate
Lathyrus & MilletYan 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
* 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;
Gene inactivation
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/CS2/1990/007http://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-new-
lathyrism-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
Eamens (2008) Plant Physiol.147:456–468
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
RNA-mediated
Mansoor et al., (2006) Trends Plant Sci. 11:559-65.
Once you know the sequence of a gene, it becomes easy to inactivate
- only that gene
- only in certain tissue/organ
Gene to be silenced
RNAi- mediated pathways in plants (2005)
Si conoscono molti geni implicati nel silenziamento