IC4R002-Metabolomics-2007-17934451

Project Title

Folate fortification of rice by metabolic engineering

The Background of This Project

• Although rice is a major staple crop, providing 80% of the daily caloric intake of 3 billion people, it is a poor source of essential micronutrients, including folates (vitamin B9). Hence, folate deficiency is widespread, especially in developing countries. Folate deficiency results in serious disorders, including neural tube defects such as spina bifida in infants and megaloblastic anemia 1,2 (Supplementary Note online). Adequate dietary folate intake can prevent onset of these conditions. Folates are tripartite molecules containing a pterin moiety, paraaminobenzoic acid (PABA) and one or several glutamate residues. In plants, pterin precursors are synthesized from GTP in the cytosol (pterin branch), whereas PABA is derived from chorismate in plastids (PABA branch). Both pterin precursors and PABA are imported into the mitochondria to participate in the condensation to folates.
• Folate biofortification of rice by means of metabolic engineering is an alternative or at least complementary solution to the existing interventions, namely industrial fortification, folate pill distribution and diet diversification 4 . Earlier this year, folate enhancement was reported in tomato 5 . This was achieved by crossing tomato plants overexpressing mammalian GTP cyclohydrolase I (GTPCHI) 6 with plants overexpressing aminodeoxychorismate synthase (ADCS) from A. thaliana (Fig. 1a), in fruits. As the activity of plant GTPCHI is presumably subject to negative feedback regulation 7 , a mammalian GTPCHI was used to avoid this negative control. Although folates in these transgenic tomatoes were enhanced up to 25-fold, levels of pterin intermediates and PABA were also elevated (>20-fold).

Plant Culture & Treatment

• Escherichia coli strains DH-5α and DB3.1TM (Invitrogen) were used for plasmid manipulations and propagation of “empty” GatewayTM vectors, respectively. Agrobacterium tumefaciens strain LBA 4404 was used for delivery of T-DNA from binary vectors into plant cells. Japonica rice (Oryza sativa L.) variety Nippon Bare plants were grown in soil under 8h of light (420 μmoles/m 2 /s light intensity, 28oC, 80% humidity) and 16h darkness (21oC, 80% humidity) regime. As a starting material for the Agrobacterium-mediated rice transformation somatic embryogenic calli were used. Empty vector transformation was performed according to Scarpella and co-workers (V) was used as a transformation control. Fifty one, 48 and 67 primary transformed lines (T0) were generated for A, G and GA constructs, respectively, in 3 transformation experiments.
  
  

Figure. 1 Engineering folate biosynthesis.

Research Findings

• The Figure 1 describes the Engineering folate biosynthesis. (a) Simplified scheme of the folate pathway in plants. Engineered enzymes are in bold. GTPCHI, GTP–cyclohydrolase I; ADCS, aminodeoxychorismate synthase; DHN-P-P-P, dihydroneopterin triphosphate; DHN, dihydroneopterin; HMDHP, hydroxymethyldihydropterine; ADC, aminodeoxychorismate; PABA, para-aminobenzoic acid. (b) Schematic representation of the T-DNA in plant transformation vectors. Open pointers depict promoters, filled pointers depict cDNA coding regions, gray bars indicate transcriptional terminators. LB and RB, left and right T-DNA borders, respectively; T35S and Tn, transcriptional terminators of the 35S transcript of cauliflower mosaic virus and nopaline synthase gene of Agrobacterium tumefaciens, respectively; 35S, Glb-1 and GluB1, core cauliflower mosaic virus 35S promoter with duplicated enhancer sequence, rice globulin and rice glutelin B1 promoters, respectively; GTPCHI, coding sequence of A. thaliana GTP cyclohydrolase I; ADCS, coding sequence of ADC synthase from A. thaliana; hptII, gene encoding hygromycin phosphotransferase (hygromycin selectable marker). (c) Expression analysis of introduced GTPCHI and ADCS genes in transgenic GA rice lines by northern hybridization using radioactive probes. Samples from seeds of homozygous T2 and T3 plants are underlined with regular and bold lines, respectively. V, control transformed with the ‘empty’ vector. Hybridization with a 25S rDNA probe was used as the loading control.

• To compare the effect of engineering the two branches of folate biosynthesis, three plant transformation vectors were constructed (Fig. 1b). The G vector contained a cDNA encoding A. thaliana GTPCHI, under the control of the rice endosperm-specific globulin (glb-1) promoter 8 . The A construct contained cDNA encoding A. thaliana ACDS under the control of the rice endosperm-specific glutelin B1 (gluB1) promoter 9 . The GA construct combined both G and A expression cassettes on a single T-DNA. All vectors were stably introduced in the genome of Nippon Bare japonica rice by Agrobacterium tumefaciens–mediated transformation. The transgenic plants had a phenotype indistinguishable from the wild type (WT) throughout development and had similar seed-set capabilities. Screening for single-copy transformation events in T0 plants and further selection of homozygous T2 individuals were done using real time PCR and Southern hybridization.

Labs working on this Project

• Unit Plant Hormone Signalling and Bio-imaging, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
• Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
• Department of Nutrition, Case Western Reserve University, 2109 Adelbert Road, Cleveland, Ohio 44106-4906, USA.

Corresponding Author

Dominique Van Der Straeten (E-mail:dominique.vanderstraeten@ugent.be)