Generation of transgenic maize with enhanced provitamin A content

Maneesha Aluru 2 Yang Xu 2 Rong Guo 2 Zhenguo Wang 1 Shanshan Li 1 Wendy White 1 Kan Wang 0 Steve Rodermel 2 0 Department of Agronomy, Iowa State University , Ames, IA 50011, USA 1 Department of Food Science and Human Nutrition, Iowa State University , Ames, IA 50011, USA 2 Department of Genetics, Development and Cell Biology , 253 Bessey Hall, Iowa State University , Ames, IA 50011, USA Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as b-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of 'Golden Rice 2' which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and z-carotene desaturase in plants), under the control of a 'super g-zein promoter' for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of b-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high b-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of bcarotene is due to an up-regulation of the endogenous lycopene b-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD. Introduction Carotenoids are C40 polyenes that are abundant in fruits, vegetables, and green plants (reviewed in Olson, 1989; Howitt and Pogson, 2006). In higher plants, all of the steps of carotenoid biosynthesis occur in plastids by enzymes that are coded for by nuclear genes and imported into the organelle post-translationally (Fig. 1) (reviewed in Hirschberg, 2001; Cunningham, 2002; Fraser and Bramley, 2004; Howitt and Pogson, 2006). The key regulatory step of the pathway is mediated by phytoene synthase (PSY) and involves the condensation of two geranylgeranyl pyrophosphate (GGPP) to form 15-cis-phytoene, a colourless C40 compound. Phytoene is converted to all-translycopene (a red pigment) by four desaturation reactions (mediated by phytoene desaturase, PDS, and f-carotene desaturase, ZDS) and by an isomerization reaction (mediated by CRTISO). Lycopene is cyclized by e and/or b-cyclase to give rise to the yellow-orange pigments, b-carotene (with two b-ionone rings) and a-carotene (with one e-ionone ring and one b-ionone ring). Alpha- and b-carotene are subsequently hydroxylated and modified to form the various xanthophylls. Carotenoids function in plant tissues as accessory pigments in photosynthesis, as attractants for seed dispersal and pollination, as precursors of some scents and of the growth regulator ABA, and as antioxidants (reviewed Fig. 1. Carotenoid biosynthetic pathway in maize. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, f-carotene desaturase; CRTISO, carotenoid isomerase; bLCY, b-cyclase; eLCY, e-cyclase; HYD, carotene hydroxylases; CRTB, bacterial homologue of PSY; CRTI, bacterial homologue of PDS and ZDS. in Hirschberg, 2001; Cunningham, 2002; Fraser and Bramley, 2004; Howitt and Pogson, 2006). Whereas carotenoid function is dependent on plastid and cell type, their role as antioxidants appears to be ubiquitous. This role is perhaps best understood in chloroplasts, where desaturated (coloured) carotenoids quench triplet chlorophyll and singlet oxygen (produced during photosynthetic light capture), preventing the formation of reactive oxygen species (ROS) and photo-oxidation of the contents of the organelle (reviewed in Niyogi, 1999). Mammals do not synthesize carotenoids de novo and thus they must be ingested in the diet. Of the ;700 carotenoids found in nature, 2050 are common in the human diet and about 20 are found in human blood and tissues (Johnson, 2004). Dietary carotenoids have received considerable attention because they have been implicated in preventing various eye and cardiovascular diseases, as well as several types of cancer and other age-related diseases, probably via their role as antioxidants and/or as regulators of the immune system (reviewed in Fraser and Bramley, 2004; Johnson, 2004). Carotenoids with unsubstituted b-ring end groups, such as a-carotene, b-carotene, and b-cryptoxanthin, have provitamin A activity. b-carotene has twice the activity of the others because it has two unsubstituted b-rings. Provitamin A carotenoids are cleaved in the intestinal lumen to produce retinal (vitamin A). The efficiency of bioconversion depends on a number of factors (e.g. the nature of the food matrix that is ingested), and bioefficacies are significantly lower in developing countries than in developed countries (West et al., 2002). Vitamin A is an essential micronutrient for human health, and the World Health Organization estimates that greater than 100 million children worldwide have vitamin A deficiency (VAD) (www.who.int/vaccines-diseases/en/vitamina/science/sci01.shtml). Nearly all of these cases are in developing countries whose populations rely on a single staple crop for their sustenance. It has been estimated that half of all VAD cases become severe and result in blindness and death. Attempts to modify the carotenoid content of seeds have focused on seed-specific manipulation of various steps in the carotenoid pathway. Overexpression of the bacterial crtB (for PSY) in the oilseeds of canola led to ;50-fold increase in total carotenoids (Shewmaker et al., 1999). These increases occurred mainly in a- and b-carotene. Using an endogenous PSY gene, enhanced seed-specific accumulation of a- and b-carotene was also achieved in Arabidopsis, but unlike canola, there was less flux into a-carotene versus other carotenoids, primarily lutein and violaxanthin (Shewmaker et al., 1999). In rice endosperm, which lacks provitamin A, overexpression of the daffodil PSY led to the production of phytoene (Burkhardt et al.,1997), but when coupled with expression of the bacterial crtI gene (which mediates the four desaturation reactions) and/or the daffodil lycopene b-cyclase gene (LCYB), there was enhanced accumulation of lutein, b-carotene, and zeaxanthin (Ye et al., 2000). These lines served as the prototype for Golden Rice (Al-Babili and Beyer, 2005). Whereas the b-cyclase gene appeared to be dispensable in these experiments, it was suggested that the source of PSY might be limiting, and thus different PSY (...truncated)