Biofortification: Engineering the metabolic pathways Swapan Datta, DDG (Criop Science), ICAR, New Delhi

EVERYTHING; THERE IS A SEQUENCE and connected to a metabolic pathway

Nutrition enriched food crop: Engineering metabolic pathways

• Importance of Nutrition Rice

• Why genetic engineering to alter the pathways?

• What and how do we understand the pathways

• Can pathways relate to functional gene expression?

• Plant breeding, Cross-talk and phenotyping

• Dream Nutrition-Rice

GLOBAL FOOD SECURITY AND MALNUTRITION

• 1.1 billion are absolutely poor with incomes < 1U$ day• 2.0 billion are marginally better off• 840 million people are food insecure• 200 million malnourished children• 400 million have acute iron deficiency• 125 million are affected by a lack of vitamin A

• Only 4% rice of the world supply is non-traded internationally

• Many of 8 billion people on the earth by 2020 will live outside the market driven supply of food

1 Billion people of world is malnourished while 30% Indian population (mostly women and children) are malnourished : Food +

Nutrition Security come together & can easily be utilized with PDS

Improved protein-potato (Ama1)Carotenoids enriched potato

Insulin promoting riceCanola with -carotene

Vitamin C food cropHigh iron rice

-carotene + Vit E riceVitamin E + -carotene maize

Biofortified food crops for India?

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Protein Calories

Nutrition from riceNutrition from rice

Lutein Zeaxanthin

GGPP -carotene biosynthesis

Pathway in transgenic rice

-carotene -carotene (3)

LC (lyc)

PDS (crt1)

PS (psy)

(1)

(2)

Phytoene

Lycopene

Vitamin EGibberellins

Chlorophyll

IPP

GGPP

Common pathway in plants (rice)

Fig. 1. Biosynthesis pathway of -carotene

Sources of Vitamin E : Tocotrienols

Primary sources of vitamin E are derived from plants. Tocopherols and Tocotrienols are plastid localised molecules.

Oil seeds are richest source of vitamin E, having total tocol levels ranging from 330 to 2,000 µg per gram. Tocotrienols are the primary form of vitamin E in seed endosperm of most monocots, including cereals, such as wheat, rice, and barley.

Tocotrienols are found in the seed endosperm of a limited number of dicots, such as tobacco and found rarely in vegetative tissues of plants

Strategies for increasing Vitamin E content in plant food

Recommended daily allowances of vitamin E is 40 I.U.

Much effort is currently aimed at identifying the genes involved in tocol biosynthesis to improve vitamin E levels in crop plants by metabolic engineering. Two strategies can be taken in this regard.

1. Produce elevated levels of total tocols through biosynthetic pathway.

2. Altering tocol composition in favor of α-tocopherol

The isolation of genes for nearly all the steps in tocopherols and tocotrienols biosynthesis has fascilitated efforts to alter metabolic flux in plant cells.

Biosynthetic pathway of Tocopherols & Tocotrienols

Vitamin E- Maize

HGGT catalyzes an analogous reaction to HPT, only it is highly specific for GGDP whereas HPT uses PDP as its prenyl substitute.

Results from the expression of barley HGGT in transgenic plants suggest that this enzyme has strong substrate specificity for geranylgeranyl diphosphate, rather than phytyl diphosphate.

Expression of HGGT enzyme in tobacco calli and Arabidopsis leaves resulted in accumulation of Vitamin E antioxidants in the form of tocotrienols ,principally as γ-Tocotrienols, and generated little or no change in the content of Tocopherols (Cahoon et al, 2003)

Barley HGGT gene was over-expressed in maize seeds, leading to a 20-fold increase in tocotrienol level, which translated to an eight-fold increase in total tocols (tocopherols and tocotrienols) (Cahoon et al, 2003).

Genotype screening for the carotenoids in brown and milled rice

Gradual Decrease of Carotenoids with the Increasing of Polishing Time (SECONDS)

AU

0.000

0.002

0.004

0.006

0.008

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0.012

Minutes

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5.00 10.00 15.00 20.00 25.00 30.00 35.00

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Genesinvolved incarotenoid

biosynthesis

Cloned/transferred

Cropspecies

Remarks Reference

Y1 cloned Maize

Importance ofsuch regulatorygene in rice is

conceptualized

Buckner et al. 1990

crtI (Phytenedesaturase)

cloned/transformed

Erwiniauredovora/Tobacco/

Herbicideresistance;Increased

Misawa et al 1990,1993

crtE cloned Erwiniaherbicola

coding for GGPPsynthase

Math et al 1992

A gene cluster clonedErwinia

herbicola

For completecarotenoid

pathwayTo et al 1994

psy transformed Tomato

Resulted indwarfism

redirecting themetabolites from

gibberellinpathway

Fray et al 1995

lcy cloned DaffodilLycopene to

beta-caroteneAl-Babili et al 1996

psycloned/

transformedDaffodil/

Rice

Accumulation ofphytoene in rice

endosperm

Scheldz et al 1996;Burkardt et al 1997

crtB(phytoenesynthase)

transformed Brassica

Overexpressionled to increasein carotenoids

and othermetabolites

Shewmaker et al1999

Selected historical developments in carotenoid metabolism in relation to plant metabolic engineering

Carotenoids biosynthesis in plants

Datta K et al (2003) Plant Biotech J (Transgenic IR64, several other cultivars using Mannose selection

system)Hoa et al (2003) Plant Physiol (Transgenic indica rice )Parkhi et al (2005) Mol Genet Genomics (Marker free BR29 GR by Agrobacterium)Paine et al (2006) Nature Biotech (High carotenoids in US cultivar)Datta K et al. (2006) Current Sci (High carotenoids indica rice)Parkhi et al (2006) Plant Sci (Protection against draught)Krishnan et al (2009) Plant Science

3.2 kb(crtI)

1.5 kb(hph)

VPBR29-9

56 59 61 64 65 66 69 70 71 72 74 1 2 3 19 27 47 51 57 NT P

VPBR29-32

Fig 3

Fig 4

VPBR29-9 VPBR29-31P NT

3.2 kb(crtI)

1.5 kb(psy)

Golden BR29 rice without a marker gene (Mol Gen Genomics 2005)

3.0-

9.1

g/g

, DH

hom

ozyg

ous

line

s de

velo

ped

Datta K et al PBJ, 2003/2005,2006Parkhi et al MGG, 2005,2006

Rai et al 2003,2006Ye et al Science, 2000

Painie et al Nature Biotech, 2005

Golden Rice (BR29) developed at IRRI is now in Bangladesh soilSyngenta-Golden Rice (GR2) is now in field at Louisiana, USA

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mm

erc

ial r

igh

t o

f G

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em

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ith

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enta

BR29

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Fig. HPLC chromatograms showing beta carotene peaks in the carotenoid extract from polished seeds of one progeny of BR29 in T1 generation

Lui

β-cry

α-crt

β-crt

BR29

Co-transformationLBA4404/pZPsC +

LBA4404/pZLcyH

Anther culture

Hemizygous T309 GoldenRice(Ye et al. 2000)

Dihaploid homozygous T309 GoldenRice(Baisakh et al. 2001b)

IR64

1st Backcrossing

F1IR64 x

IR64BC1F1

x

x

2nd Backcrossing

BC2F1

Marker-free

SelfingBC2F2

Marker-free

PCR analysis

Molecular analysisPhenotyping

Molecular analysis

Selection of hph negative transgenic progenies

PCR screeningand Southern confirmation

IR64 NILsMarker-free

Phenotyping HPLC

BC1F1 progeniesMarker-free

Flow chart for the

Development of

Marker-free

Near-isogenic golden

Rice lines of IR64

CharactersTreatments

Plant height (cm)

No. of panicles per plant

No. of grains per panicle

No. ofunfilled spikelets per panicle

Spikelet fertility(%)

1,000- grain weight (g)

Biological yield per plant (g)

Grain yield per plant (g)

Harvest index (%)

TRANSGENIC

Mean 107.13 9.13 88.81 34.16 71.46 25.86 109.25 13.49 13.66

SEm 0.745 0.358 2.460 1.364 1.078 0.168 5.953 0.661 0.610

CONTROL

Mean 108.80 8.65 86.05 28.75 74.67 25.77 98.98 13.74 14.86

SEm 1.733 0.539 5.558 3.312 2.635 0.223 9.309 1.350 1.290

F-value(transgenic vs. control)

0.950ns 0.391ns 0.242ns 2.881ns 1.627 ns 0.060ns 0.702 ns 0.030ns 0.770 ns

Agronomic performance of transgenic Golden rice (cv. IR64) vis-à-vis the IR64 control

ns= nonsignificant at p 0.05 (Rai et al. RGN 2004)

Fig. 3. Transgenic Golden indica rice of NHCD (lanes 1 and 2 in each panel) and IR64 (lanes 4, 5, 6, and 7 in each panel) showing no polymorphism with Universal rice primers (URP) vis-à-vis their respective controls (lanes 3 and 8 in each panel). M = 1 kb-plus molecular weight marker.

Fig. 1. Southern blot showing homozygous progenies of Golden indica rice (cv. IR64) with integration of a 3.8-kb fragment

12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M 12 3 4 5 6 7 8 M12 3 4 5 6 7 8 M M1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8 M

URP1 URP2 URP3 URP4 URP5 URP6 URP7 URP8 URP10 URP11

Fig. 2. Transgenic Golden indica rice (T) and control rice (cv. IR64; C) showing uniformity in overall phenotype (left panel) and grain filling (right panel) grown under screenhouse conditions at IRRI, Philippines.

T C C T

NT PCT3 progenies of transgenic golden IR64

3.8-kb

Essential Minerals: IronIron deficiency is the most widespread micronutrient

deficiency worldwide.

Approx. 30% of world population suffers from serious nutritional problems caused by insufficient intake of iron (WHO 1992).

It is the important constituent of hemoglobin, the oxygen carrying component of blood, and also a part of myoglobin that helps muscle cells to store oxygen.

It is present in food in both inorganic (ferric and ferrous) and organic (heme and nonheme) forms. Highly bioavailable heme iron is derived primarily from animal source.

Biofortified iron rice

1. High iron and enhanced carotenoids/beta-carotene rice 2. Reduced content of phytate in rice grains

Mutationalbreeding

Transgenic plantstrategy

Screening for iron-rich rice varieties

Increased bioavailabillity of Fe and Zn

ferritin 35S g7barGluB-1nos

Sst I Bam HI Hind III

ferritin Glo-Pnos

Sst I Bam HI Kpn I

ferritin Pro-Pnos

Sst I Bam HI Kpn I

Vasconcelos et al Plant Sci 2003Tan et al Int J Food Sci Tech 2004

Khalekuzzaman et al In J Biotech 2006

The Aspartate-Family Biosynthetic Pathway

Aspartate

-aspartyl phosphate

aspartic -semialdehyde

AK

2-3 dihydropicicolinate5 steps

Threonine MethionineLysine

DHDPS

Technologies Ready for transfer

1. 30 Normal and 8 QPM SCH

2. Baby corn, Sweet corn, popcorn single cross hybrids available

3. Technology for Single Cross Hybrid Seed Production and commercial cultivation for normal QPM and specialty corn

Sweet corn hybrid HSC-1

Pop corn hybrid Hyd 14-3 X

HKIPC5

Normal maize

SCH Seed production

Q PM

Value added Dream-RICE

• High iron rice (after polishing)

Provitamin A rice

Other micronutrient-rich rice

Development of Value added rice for both favorable and unfavorable ecosystems. combination of high yield with value-added rice

Green revolution saved famine in Asia

Molecular breeding for Nutrition food may help in reducing malnutrition

provided FTO (Govt supp.) is in place