Biofortification: Plant Breeding to Solve World-wide Micronutrient Malnutrition “Golden rice”, rice that is enriched in pro-vitamin A, is the “poster child”

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Biofortification:Plant Breeding to Solve

World-wide Micronutrient Malnutrition

“Golden rice”, rice that is enriched in pro-vitamin A,is the “poster child” for bio-fortification. The Bill and Melinda Gates Foundation has identified micronutrient malnutrition as one of the worldwide problems they want to help and solve and are contributing millions of $$ for research.

Grains are deficient in vitamins C, B12, A, E and calcium



A cereal-based diet is poor in specific micronutrients. The presence of 21 nutrients in 19 averaged grains, when

they supply 2200 calories in the diet. Grains are deficient in Vitamins A, E, C, B12 and calcium

RDA = recommended daily average



Vegetables are rich in micronutrients.The presence of 21 nutrients in an average of 96 vegetables

when they supply 2200 calories. Only vitamin B12 is missing.Notice 10 times greater cost.

Micronutrient deficiency is a world-wide problem.



Iron deficiency

Iron and vitamin A deficiency

Iron, iodine and vitamin A deficiency

What is the scope of the problem?

Vitamin A deficiency: 3 million children have visible eye damage. Sub-clinical prevalence is probably 100-200 million. 300,000 children go blind every year and 2/3 of them die within months of going blind.

Iron deficiency: 3 billion people are iron deficient. Caused by low iron intake and blood loss (intestinal parasites)

Zinc deficiency: extent is unknown; clinical symptoms are unclear; poor hair growth is one symptom.

Iodine deficiency: 2 billion people live in iodine deficient areas; It is the greatest single preventable cause of brain damage and mental retardation. It is the leading cause ofintellectual impairment in the world.

Sometimes, the only way to ascertain that deficiencies exist is to supplement the diet. In many cases there are no clinical manifestations.

Mild iodine deficiencycauses goiter, hypertrophy

of the thyroid gland.

Why bio-fortification and not just food fortification or vitamin pills?QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture. QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

Bio-fortification is self-perpetuating. Once the plants have been Created the farmers can keep on planting them.

Effective in remote rural areas. Seeds can be distributed to rural areas. There is no dramatic change in food habit, or need to remember to take a pill.

Not dependent on political forces/budgets. Distribution of vitaminpills has to be authorized/paid for by international organizations or governments. Many Developing countries lack the infrastructure for distribution.

More cost effective by a factor of 10 (50 M versus 500 M)Cost of supplementation is continuous and cost of creating the plants is one-time.

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Functional foodsA functional food is any food or food ingredients that provides a health benefit beyond the traditional nutrients it contains. May

contain food additives, dietary supplements, probiotics or medical foods.

• Monsanto Vistive 1 On the market; low linolenic acid soybean oil; reduces the need for hydrogenation and contains less trans fatty acids (TFAs).

• Dupont High Oleic GM soybean ; almost through regulatory; stable for frying; like olive oil. Similar to Vistive 1, but GM crop.

• Danone Activia yoghurt; commercial success; with Bifido bacteria that improve regularity. A probiotic food.

• Olivenol, a CreAgri product extracted from olive oil; contains polyphenol hydroxyltyrosol an antioxidant; a dietary supplement.

• Efficas care product marketed by Efficas contains a proprietary ratio of -linolenic acid and eicosapentanoic acid to reduce inflamation associated with allergies and eczema. A medical food

• Attune wellness bars with 3 strains of bacteria that support digestion and promote immunity

“Golden Rice” (rice enriched in -carotene or pro-vitamin A) is the paradigm for the international bio-fortification

effort.Unfortunately, golden rice is controversial because it is genetically

modified. Biofortification is also proceeding with traditional breeding

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Prof. Ingo Potrykus from theETH in Zurich, Switzerland.

Photosynthetic organisms synthesize carotenoids to protect the photosynthetic apparatus from light overload.

Carotenoids and xanthophylls are lipid pigments that accumulate in chloroplasts (that also have chlorophyll) and chromoplasts (that don’t have chlorophyll) and give yellow and red colors to tomatoes, marigolds, carrots etc. Chloroplasts and chromoplasts are

convertible (e.g.when tomatoes become red the chlorophyll disappears and carotenoids are synthesized. Carotenoids are also made by green algae









Note thylakoids (flat membranes) and lipid globules

chromoplast

In fruits, the function of carotenoids isprobably seed dispersal by animals

Carotenoids are a family of structurally related lipids (more than 100) synthesized by plants, algae and bacteria.

Some are polar, others are not. Polar ones have greater antioxidant function, resulting from their positioning in membranes, but are not vitamin A precursors.

Bioavailability of -caroteneand conversion to vitamin A.

Bioavailability relates to the fraction of carotene that the body can extract out of a given foodstuff and and use to make vitamin A; it is determined to a great extent by the food matrix, however, within a plant tissue, carotenoids can be stored in several ways:

* Protein-bound, stoichiometrically (in light-harvesting complexes, in green vegetables); also in lobsters. * Protein-associated; by a protein that organizes a proteolipid-carotenoid complex with a protein called fibrillin and homologs thereof, as in red pepper. * Free in membranes; in chromoplasts, like in daffodil (probably etioplasts and amyloplasts). * In plastoglobules, frequently found in flowers, algae (probably the most bioavailable). * In crystals, as in tomato and carrot (this is probably the least available form). * Additional determinants may act as promoters or inhibitors of resorption.

This means that a person can in principle obtain more utilisable ß-carotene from an orange-fleshed sweet potato which has ca. 18 µg/g ß-carotene than from carrots that have around 60 µg/g.

RDA for vitamin A is 700 - 900 g/day

Vitamin A is trans-retinol. Retinal is made from -carotene (pro-vitamin A) by a single enzymatic step by a

dioxygenase in the intestinal mucosa. Retinal is reduced by NADH and immediately esterified with palmitic acid. This ester is stored in the liver and

released when needed. It functions in vision and reproduction. Adults need 0.7 mg per day. Night blindness is one of the first symptoms of vitamin A deficiency.



retinal

Carotenoids and human health: important for vision, for embryogenesis and as antioxidants.

Carotenoids are 20-carbon polyprenols and are important anti-oxidants because of their system of conjugated bonds.

The carotenoids lutein and zeaxanthin are concentrated in the central retina, overlying the macula, a small spot where color vision is produced.

-carotene (pro-vitamin A) is converted to retinal which is covalently bound to

the protein opsin in the rod and cone cells of the retina.



The cis/trans isomerization of retinal is the basis of vision and starts the signal

transduction cascade .



Change in retinal causes a conformational change in the protein opsin, a G-protein coupled receptor to which retinal is covalently bound.

When double bonds alternate with single bonds, they are termed "conjugated”. This means that the electrons that make up the double bonds in the linear chain are "delocalized", or shared evenly over the whole chain. This makes the whole chain relatively electron-rich. Conjugated double bonds are very chemically stable, yet are capable of specific chemical reactions that require their electron-rich yet stable structure. For example, if a carotenoid loses one electron and becomes a carotenoid cation (positively charged ion), the resulting

charge of +1 is distributed over the electron-rich chain, a much more stable situation than if the charge were limited to a single location on the compound.

In mammalian cells, carotenoids protect from oxidative damage by two general mechanisms:* quenching of singlet oxygen and dissipating the energy as heat* scavenging of radicals to prevent or terminate chain reactions.

Singlet oxygen is a more reactive state of molecular oxygen in which all the electron spins are paired.This oxygen species is more reactive towards other macromolecules, oxidizing them.

Carotenoids as antioxidants

Retinoic acid activates a number of genetic pathways important for normal embyonic development





Development of primary neurons inXenopus laevis development.a: controlb: excess retinoic acid

c: deficiency in retinoic acid The tissue-specific location and timing of retinoic acid production during embryogenesis provides signaling information needed to generate the central nervous system, heart, eyes, vertebrae, and limb buds.

Carotenoids are isoprenoids or polyprenols.The bioynthesis of all polyprenols starts with the synthesis of isopentenyl-pyrophosphate (IPP). Plants have 2 pathways to make IPP: the eukaryotic

(Mevalonate or MVA) pathway and the prokaryotic (Methyl erythritol phosphate MEP) pathway in the chloroplasts. Different isoprenoids with

quite different functions are made by these two pathways.

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PolyprenolsPolyprenols are formed by the

multistep condensation of the C5 building blocks, first to C10

(geranylpyrophosphate or GPP), then to C15 (farnesylpyrophosphate

or FPP), then to C20 (geranyl geranyl pyrophosphate or GGPP) and finally condensation of two GGPP molecules by phytoene

synthase. The first C40 product, phytoene, is colorless.

A knock-out (gene inactivation) of phytoene synthase produces

chlorotic (white) plants.Carotenoids are essential to protect chlrophyll and the photosynthetic

apparatus from photooxidative damage (xanthophyll cycle)

C10

C15

C20

C40

Pro-vitamin A

Phytoene to lycopene:four desaturationsteps carriedout by one bacterial phytoene desaturase or by two different plantdesaturases: phytoenedesaturase and zeta-carotene desaturase

**

**

GGDP

Phytoene

Phytofluene

-carotene

-carotene-carotene

Lycopene

Neurosporene

cyclases

Zeta carotene

Beta ring Gamma ring Epsilon ring

carotene was crystallized in 1831 and named after the source: Daucus carota.The carotenes were named in the order that they were discovered. Compoundsthat have a ring can give rise to vitamin A (retinol)

Lutein

-carotene

zeaxanthin

anteraxanthin

violaxanthin

neoxanthin

Xanthophylls and lutein haveoxygen atoms in their rings.Their synthesis requires hydroxylases and epoxidases.

Pro-vitamin A

Strategy for making -carotene in rice endosperm. Rice endosperm can make geranyl geranyl pyrophosphate, but

then you need phytoene synthase to make phytoene, the colorless precursor of carotenoids. Two desaturases, phytoene

desaturase and -carotene desaturase, are needed to introduce two double bonds each, followed by lycopene -cyclase to make

-carotene. The two desaturases can be replaced by a single bacterial enzyme. All enzymes must have plastid transit

sequences and all genes must/should have endosperm specific promoters. Ye et al, Science, 287: 303-305. 2000

“Golden Rice”Golden RiceSee also: http://www.goldenrice.org/

This vector has two genes: the Narcissus phytoene synthase (psy) is driven by theglutelin promoter and with the nos! terminator. It does not need an extra Chloroplast transit sequence because this is a nuclear gene and the protein is normally made with the transit sequence (tp); the Erwinia uredovora phytoene desaturase (crtl) is driven by the CaMV 35 S promoter and has the nos! terminator. The selectable marker is the aphIV gene and selection is on hygromycin.

This vector has the third gene: it encodes the Narcissus lycopene b-cyclase and isalso driven by the glutelin promoter and has the 35 S! terminator. It does not needa transit sequence (see above)

HPLC chromatography of carotenoids in endosperm of

transformed rice. A. Control

B. transformed with phytoene synthase and bacterial desaturase on a single vector; these two genes should

direct the synthesis of lutein and zeaxanthin from GGPP;(there

is also some carotene made apparently) C. double transformants using both vectors at the same

time; these three genes should increase -carotene synthesis (cyclase) . Arrow shows position of lycopene.

Control

Phenotypes of seeds from plants expressing all three genes. 1-4 represent different lines and show the variability in initial transformants. The best lines produce 1.6 g of -carotene per gram of rice, for a daily intake of 0.5 mg assuming you eat 300 g of rice). Bioavailability?

Greenpeace calculations show that an adult would have to eat at least 3.7 kilos of dry weight rice, i.e. around 9 kilos of cooked rice, to satisfy his/her daily need of vitamin A

from "Golden Rice". In other words, a normal daily intake of 300 gram of rice would, at best, provide 8% percent of

the vitamin A needed daily. A breast-feeding woman would have to eat at least 6.3 kilos in dry weight,

converting to nearly 18 kilos of cooked rice per day.


are needed to see this picture.The Position

The recommended daily allowance for Vitamin A is 700 g per day for females and 900 g per day for males. Your intake of carotene possibly needs to be 10 x as much because of inefficient conversion. The calculation is correct but the assumptions are not!Incorrect assumptions: (1) This is the best rice we can make; (2) You need 700 mgper day to avoid blindness; (3) the diet of poor people has no other source of Vit A.

Lines used for subsequent calculation.

SGR 1 SGR2Wildtype

SGR2: 16g developed by private sector; donated to the humanitarian

project

SGR1: 1.6g regulatory

clean; jointly developed by

public & private sector.

How much rice does a child need to eat to prevent VADD?

Calculation by the International Food Policy Institute:

How much rice does a child have to eat to prevent VADD?

Vitamin A in fish &

animal food

Provitamin A in vegegables

and fruit

Even poor Bangladeshis eat not just rice and will get beta-carotene from other sources

50% RDA is sufficient to prevent vitamin A malnutrition!

How much rice does a child need to eat to prevent VADD?

Estimation from International Food Policy Institute:

(2) Vitamin A contribution from food intake.

Conversion factor used: 12:1

RDA

VADD without Golden Rice!

No VADD with Golden Rice!

Is Golden Rice safe to eat?Most likely, but feeding tests need to be done.

The human body regulates the conversion of carotene to retinol and only makes as much as is needed. Excess is stored as ester in the liver.

Field tests are now in progress to harvest enough rice for human feeding tests.

What about bioavailability?Not yet determined. Is carotene in plastoglobuli or crystalloids?

Will golden rice solve the vitamin A deficiency problem?Probably not right away. Food and nutrient shortages are very much related to

poverty - lack of purchasing power - and to other societal factors. Getting new cultivars to the farmers who need them is extremely difficult.

Furthermore, rice is not a staple for everyone.

The Greenpeace position against field release experiments with Golden Rice is a fake argument to save a radical position. The moral challenge gains additional weight because Greenpeace has, so far, not presented any concrete scenario for an environmental hazard from Golden Rice beyond the unsubstantiated notion

that "release of transgenic plants into the environment poses an unacceptable risk to the environment". In view of the foreseeable benefit from Golden Rice,

Greenpeace should be expected to be a bit more concrete with its environmental argument.

What is the risk from Golden Rice, which has no selective advantage in whatever environment, and which produces just a few micrograms more of an environmental neutral substance (-carotene) in the endosperm, in addition to

the same substance being present in large quantities in all other parts of the natural plant? How does such a (so far undefined, hypothetical) risk compare to the expected benefit? We believe that the public has a right to a more concrete

answer from Greenpeace.

Statement by Ingo Potrykus and Peter Beyer

Is Golden rice safe for the environment?

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers. G. Giuliano laboratory

Potatoes have no -carotene; yellow color is due to xanthophylls: lutein and violaxanthin; these haveno pro-vitamin A activity; antisense of LCY-e directs flux towards -carotene, raising -carotene 10-fold; silencing of CHY raises -carotene; color indicates x increase.

lutein

violaxanthin

-carotene

X

X

Orange cauliflower: the or (orange) mutation activates chromoplast development and carotene biosynthesis.

Brassica oleracea var botrytis

OR is a DNA J protein (chaperone) with a transit peptide. The gene is expressed highly in tissues that have

proplastids or leucoplasts. It is a negative regulator of chromoplast development. When mutated chromoplast development from proplastids is allowed to proceed.

White cauliflowers transformed with Or become orange.

Can we find such a mutation in rice endosperm?



Transformation of potatoLu et al, The Plant Cell 18:3594-3605 (2006)

Or-Gus fusion

*or mutant

Golden corn?

Means and Ranges from 2002 Survey Diverse Genetic Set of Inbreds

_-carotene 1.31 0.06 - 5.93

_-carotene 0.26 0.07 - 0.91

_-cryptoxanthin 1.17 0.14 - 4.47

Lutein 9.53 0.36 - 32.39

Zeaxanthin 7.47 0.49 - 28.49

Total Carotenoids 19.78 0.98 - 49.94

Variable Mean (_g g -1) Range (_g g -1)

-Mean Two Replicates 85 of 102 Inbreds Able to Produce Seed

II III IV V VI VII IX XVIII

3.06

5.01

10.07

I

5.07

8.05

2.0

2.02

5.05HYD

2.09

04

4.08

9.01

Y1

ZDS EC

WC

BC = LCYB= Lycopene Beta Cyclase

HYD

BC

DXS

The level of LCYE determines the flux to or carotene. Four polymorphisms of the lcyE gene explain 58 % of the variability and a 3-fold difference in provitamin A compounds (-carotene, -carotene and -cryptoxanthin)

Carotenoid Biosynthetic Pathway

ZDSZDS

GGPPGGPP

PhytoenePhytoene

PhytofluenePhytofluene

- carotene- carotene

NeurosporeneNeurosporene

PSY PSY

PDSPDS

LycopeneLycopene

- carotene- carotene -carotene-carotene

-crytptoxanthin

ZeaxanthinLutein

- LCY- LCY- LCY- LCY

- LCY- LCY

vp9vp9

vp5vp5

y1y1

2x

2x * **

Desaturases

Cyclases

“Hydroxylases”

*

*

Synthase

Certain pearl millet cultivars have as much pro-vitamin A as

Golden Rice. So, is Greenpeace right after all and we don’t need Golden Rice?

The problem is that corn eaters like corn and rice eaters like rice and millet

eaters like millet!

Bio-accumulation of carotenoids in natural and commercial food chain.

Astaxanthin is essential for shrimp health

Commercial production of astaxanthin inthe microalga Hematococcus pluvialis

Expression of carotenoid genesin soybean seeds.

The color of tuna fish filet depends on the amount of carotenoids in the diet. Cost of pigments in the diet can be 10 % of the total fish feed cost.

-Carotene is heat and light labile

Documents

Biofortification: Plant Breeding to Solve World-wide Micronutrient Malnutrition “Golden rice”, rice that is enriched in pro-vitamin A, is the “poster child”