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 rice Canola with  -carotene Vitamin C food crop High iron rice  -carotene + Vit E rice Vitamin E +  -carotene maize Biofortified food crops for India?

Nutrition 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 E Gibberellins 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).

Vitamin E enriched corn Genetic change resulting from crop domestn. took 10,000 years. Teosinte (L) and corn or maize(R)

Genotype screening for the carotenoids in brown and milled rice

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

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 ) VPBR NT P VPBR29-32 Fig 3 Fig 4 VPBR29-9 VPBR29-31 P NT 3.2 kb ( crtI ) 1.5 kb ( psy ) Golden BR29 rice without a marker gene (Mol Gen Genomics 2005)

 g/g, DH homozygous lines developed Datta K et al PBJ, 2003/2005,2006 Parkhi et al MGG, 2005,2006 Rai et al 2003,2006 Ye et al Science, 2000 Painie et al Nature Biotech, 2005 Golden Rice (BR29) developed at IRRI is now in Bangladesh soil Syngenta-Golden Rice (GR2) is now in field at Louisiana, USA Commercial right of GR remains with Syngenta

BR29

Fig. HPLC chromatograms showing beta carotene peaks in the carotenoid extract from polished seeds of one progeny of BR29 in T 1 generation Lui β-cry α-crt β-crt BR29

Co-transformation LBA4404/pZPsC + LBA4404/pZLcyH Anther culture Hemizygous T309 GoldenRice (Ye et al. 2000) Dihaploid homozygous T309 GoldenRice (Baisakh et al. 2001b) IR64 1 st Backcrossing F1F1 IR64 x BC 1 F 1 x x 2 nd Backcrossing BC 2 F 1 Marker-free Selfing BC 2 F 2 Marker-free PCR analysis Molecular analysis Phenotyping Molecular analysis Selection of hph negative transgenic progenies PCR screening and Southern confirmation IR64 NILs Marker-free Phenotyping HPLC BC 1 F 1 progenies Marker-free Flow chart for the Development of Marker-free Near-isogenic golden Rice lines of IR64

Characters Treatments Plant height (cm) No. of panicles per plant No. of grains per panicle No. of unfilled spikelets per panicle Spikelet fertility (%) 1,000- grain weight (g) Biological yield per plant (g) Grain yield per plant (g) Harvest index (%) TRANSGENIC Mean SEm  CONTROL Mean SEm  F-value (transgenic vs. control) ns ns ns ns ns ns ns ns 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 M M M 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. TC C T NT PC T 3 progenies of transgenic golden IR kb

Essential Minerals: Iron  Iron 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 Mutational breeding Transgenic plant strategy Screening for iron- rich rice varieties Increased bioavailabillity of Fe and Zn

ferritin 35S g7 barGluB-1nos Sst I Bam HIHind III ferritinGlo-Pnos Sst I Bam HIKpn I ferritinPro-Pnos Sst I Bam HIKpn I Vasconcelos et al Plant Sci 2003 Tan 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 dihydropicicolinate 5 steps Threonine Methionine Lysine 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