Review article Biofortification of plants with altered antioxidant content and composition: genetic engineering strategies Changfu Zhu 1,† , Georgina Sanahuja 1,† , Dawei Yuan 1 , Gemma Farre ´ 1 , Gemma Arjo ´ 1,2 , Judit Berman 1 , Uxue Zorrilla- Lo ´ pez 1 , Raviraj Banakar 1 , Chao Bai 1 , Eduard Pe ´ rez-Massot 1 , Ludovic Bassie 1 , Teresa Capell 1 and Paul Christou 1,3, * 1 Departament de Produccio ´ Vegetal i Cie `ncia Forestal, Universitat de Lleida-Agrotecnio Center, Lleida, Spain 2 Departament de Medicina, Universitat de Lleida-Institut de Recerca Biome `dica de Lleida (IRBLleida), Lleida, Spain 3 Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats, Passeig Lluı´s Companys, Barcelona, Spain Received 20 June 2012; revised 4 August 2012; accepted 8 August 2012. *Correspondence (Tel +34 973702693; fax +34 973238264; email christou@pvcf.udl.es) † These authors contributed equally to this work. Keywords: biofortification, antioxidants, genetic engineering. Summary Antioxidants are protective molecules that neutralize reactive oxygen species and prevent oxidative damage to cellular components such as membranes, proteins and nucleic acids, therefore reducing the rate of cell death and hence the effects of ageing and ageing-related diseases. The fortification of food with antioxidants represents an overlap between two diverse environments, namely fortification of staple foods with essential nutrients that happen to have antioxidant properties (e.g. vitamins C and E) and the fortification of luxury foods with health- promoting but non-essential antioxidants such as flavonoids as part of the nutraceuticals/ functional foods industry. Although processed foods can be artificially fortified with vitamins, minerals and nutraceuticals, a more sustainable approach is to introduce the traits for such health-promoting compounds at source, an approach known as biofortification. Regardless of the target compound, the same challenges arise when considering the biofortification of plants with antioxidants, that is the need to modulate endogenous metabolic pathways to increase the production of specific antioxidants without affecting plant growth and development and without collateral effects on other metabolic pathways. These challenges become even more intricate as we move from the engineering of individual pathways to several pathways simultaneously. In this review, we consider the state of the art in antioxidant biofortification and discuss the challenges that remain to be overcome in the development of nutritionally complete and health-promoting functional foods. Introduction Fortification is the process of adding essential micronutrients and other health-promoting compounds to foods. The fortification of processed foods such as flour, bread, packaged cereals, dairy products and salt is a public health policy in the industrialized world, aiming to reduce the number of people suffering from malnutrition and to increase general health and wellbeing in the population. In developing countries, fortification programmes are often unsustainable due to poor governance, inefficient food- distribution networks and the prevalence of subsistence agricul- ture in rural populations, which means that most agricultural products are not processed centrally before distribution and consumption. Approximately 50% of the global population is thought to be malnourished but the vast majority of malnour- ished people are the rural poor in developing countries, subsisting on a diet of milled cereal grains lacking many essential nutrients and other health-promoting compounds (Farre ´ et al., 2011a,b; Yuan et al., 2011). In these settings, biofortification is a more sustainable strategy because this involves the fortification of crops at source either through the application of nutrient-rich fertilizers or the breeding or engineering of crops to synthesize and/or accumulate nutritionally important compounds, therefore avoid- ing the need to fortify processed food products (Go ´ mez-Galera et al., 2010). Biofortification programmes generally focus on essential micronutrients, which are either organic compounds (vitamins) or minerals required in amounts <1 mg/day. These compounds act as cofactors or metabolic precursors and are required for specific biological processes, such that insufficient intake results in characteristic deficiency diseases (Zhu et al., 2007; Go ´ mez-Galera et al., 2010; Table S1). The major deficiency diseases in devel- oping countries correspond to essential nutrients that tend to be present at low levels in milled cereal grains, for example vitamin A, iron, iodine, zinc, vitamin C and folic acid. As well as their requirement for particular metabolic processes, certain essential nutrients also act as antioxidants or promote the activity or availability of antioxidants, which help to prevent diseases that result from or that are exacerbated by the accumulation of oxidative damage to cells, including cancer, cardiovascular disease and neurodegenerative disorders. Many non-essential molecules consumed in the diet are also antioxidants with health- promoting effects, and hence there is an overlap between essential nutrients and non-essential compounds (sometimes described as nutraceuticals) that act as antioxidants. A key example of such a ‘dual-purpose nutrient’ is vitamin A, which is obtained in the diet either as esters of retinol from meat and dairy products or as pro-vitamin A carotenoids such as b–carotene from plants. Vitamin A is converted into the visual pigment rhodopsin (retinal), in the retina of the eye, and acts as a ª 2012 The Authors Plant Biotechnology Journal ª 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd 129 Plant Biotechnology Journal (2013) 11, pp. 129–141 doi: 10.1111/j.1467-7652.2012.00740.x