Folate biofortification in food plants Samir Bekaert 1 , Sergei Storozhenko 1 , Payam Mehrshahi 2 , Malcolm J. Bennett 2 , Willy Lambert 3 , Jesse F. Gregory III 4 , Karel Schubert 5 , Jeroen Hugenholtz 6 , Dominique Van Der Straeten 1 and Andrew D. Hanson 7 1 Unit Plant Hormone Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 2 Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK, LE12 5RD 3 Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium 4 Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611, USA 5 Biology Department, Washington University, St. Louis, MO 63130, USA 6 Wageningen Center for Food Sciences, PO Box 557, 6700 AN Wageningen, The Netherlands 7 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA Folate deficiency is a global health problem affecting many people in the developing and developed world. Current interventions (industrial food fortification and supplementation by folic acid pills) are effective if they can be used but might not be possible in less developed countries. Recent advances demonstrate that folate bio- fortification of food crops is now a feasible complemen- tary strategy to fight folate deficiency worldwide. The genes and enzymes of folate synthesis are sufficiently understood to enable metabolic engineering of the path- way, and results from pilot engineering studies in plants (and bacteria) are encouraging. Here, we review the cur- rent status of investigations in the field of folate enhance- ment on the eve of a new era in food fortification. Folates as vitamins and the need for biofortification Folate is a generic term for tetrahydrofolate (THF) and its derivatives (Figure 1). Folates are B vitamins, necessary in almost all organisms as cofactors for one-carbon (C 1 ) trans- fer reactions, generally referred to as C 1 -metabolism. Vitally important aspects of C 1 -metabolism are nucleotide biosynthesis, amino acid metabolism and the methylation cycle, which supplies numerous methylation reactions with methyl groups (for reviews see Refs [1–3]). Humans and other animals cannot synthesize folates and, therefore, need them in the diet, with plants usually being the main dietary sources [2]. Folate levels vary among food plants; the cereal staples maize, wheat and, particularly, rice contain extre- mely low levels (USDA National Nutrient Database for Standard Reference. Release 19; http://www.nal.usda.gov/ fnic/foodcomp/search/)(Table 1). Reliance on such staples cannot satisfy recommended dietary allowances (RDA), set at 400 mg of dietary folate equivalents (DFE) day À1 for adults National Institutes of Health Office of Dietary Supplements Dietary Supplement Fact Sheet: Folate; http://ods.od.nih.gov/factsheets/folate.asp). Clinical and epi- demiological evidence shows that folate intake is suboptimal for most populations in developing countries – as well as for surprisingly large population groups in developed countries [4–6]. Suboptimal folate intake perturbs C 1 -metabolism, which contributes to megaloblastic anemia, birth defects [neural tube defects (NTD)], and increased risks for cardi- ovascular disease and certain cancers (Box 1) [7]. Folate deficiency is, therefore, a global health problem. Although fortification and supplementation (vitamin pills) are effective ways to improve folate status, they remain far from accessible to the poor, rural population in developing countries [8,9]. Hence, there is a compelling case for the development of folate-enriched food plants as a sustain- able complement to the existing interventions for fighting folate deficiency [9–11]. Recently, major progress has been achieved not only in our understanding of the regulation of the folate biosynthesis pathway, but also in establishing a proof of concept for folate biofortification by metabolic engin- eering of crops plants. Here, we discuss these achievements, after providing background on the biochemical processes that affect folate content. Research on bacterial and animal systems is included where relevant. Folate biosynthesis and transport The steps in folate synthesis are the same in plants and bacteria, and the pathway enzymes and their genes are all known in both groups [12,13]. In essence, the three parts of the THF molecule – the pteridine, p-aminobenzoate (p- ABA) and glutamate moieties (Figure 1) – are produced separately and then joined together. In bacteria, the whole process takes place in the cytosol, but in plants three subcellular compartments are involved: plastids, mito- chondria and cytosol [14] (Figure 2). The pteridine moiety is formed from guanosine triphosphate (GTP) in the cytosol and the p-ABA moiety is formed from chorismate in plas- tids. Pteridine and p-ABA are then transported to the mitochondria, where they are coupled together, glutamy- lated and reduced to produce THF. A short chain of g-linked glutamates can then be added in mitochondria, plastids or cytosol, yielding folate polyglutamates (THF- Glu n ). Folate molecules exist in vivo mainly as polygluta- mates and these are preferred by folate-dependent enzymes involved in C 1 -metabolism [15]. By contrast, Review Corresponding authors: Van Der Straeten, D. (dominique.vanderstraeten@ugent.be); Hanson, A.D. (adha@ufl.edu). 28 1360-1385/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2007.11.001 Available online 20 December 2007