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)

http://www.poppyseedtea.com/ Poppy seed tea can kill you

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&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-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