Metabolite Sorting of a Germplasm Collection Reveals the Hydroxylase3 Locus as a New Target for Maize Provitamin A Biofortification      

Abstract

Vitamin A deficiency, a global health burden, can be alleviated through provitamin A carotenoid biofortification of major crop staples such as maize (Zea mays) and other grasses in the Poaceae. If regulation of carotenoid biosynthesis was better understood, enhancement could be controlled by limiting b-carotene hydroxylation to compounds with lower or no nonprovitamin A activity. Natural maize genetic diversity enabled identification of hydroxylation genes associated with reduced endosperm provitamin A content. A novel approach was used to capture the genetic and biochemical diversity of a large germplasm collection, representing 80% of maize genetic diversity, without having to sample the entire collection. Metabolite data sorting was applied to select a 10-line genetically diverse subset representing biochemical extremes for maize kernel carotenoids. Transcript profiling led to discovery of the Hydroxylase3 locus that coincidently mapped to a carotene quantitative trait locus, thereby prompting investigation of allelic variation in a broader collection. Three natural alleles in 51 maize lines explained 78% of variation and approximately 11-fold difference in b-carotene relative to b-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of b-carotene. A simple PCR assay to track and identify Hydroxylase3 alleles will be valuable for predicting nutritional content in genetically diverse cultivars found worldwide.

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

“…This intron-exon organization is also consistent with the maize β-hydroxylase genes (Vallabhaneni et al, 2009). The Arabidopsis β-hydroxylases, on the other hand, contain seven exons and six introns (Tian et al, 2003).…”

“…1c). Among monocotyledonous plants, two β-hydroxylase homologs have been identified from diploid sorghum (Vallabhaneni et al, 2009), four from ancient tetraploid maize (Vallabhaneni et al, 2009;Yan et al, 2010), and six from hexaploid wheat (this study), suggesting that a single duplication of the ancestral β-hydroxylase gene occurred before the divergence of the grass subfamilies (Fig. 1c, bootstrap confidence 100%).…”

“…These TCs and ESTs were further assembled into two paralogous genes, each with three homeologs, which encode proteins that are 72% identical to each other over 90% of the protein length. Several acronyms have been used in the literature for β-hydroxylases cloned from different plant species, including BCH in Arabidopsis, CrtR-b in tomato, CHY in potato, BCH and HYD in rice, CrtR-B and HYD in maize, and HYD in sorghum (Diretto et al, 2007;Du et al, 2010;Galpaz et al, 2006;Kim et al, 2009;Tian et al, 2003;Vallabhaneni et al, 2009;Yan et al, 2010). To avoid further complications in β-hydroxylase nomenclature, we designated the two newly cloned wheat β-hydroxylases as HYD1 and HYD2, to be consistent with the majority of their monocotyledonous homologs (Vallabhaneni et al, 2009).…”

“…Among monocotyledonous plants, between two to four β-hydroxylase genes have been tentatively identified from sorghum, rice, and maize based on sequence homology to known β-hydroxylases. However, only two maize β-hydroxylases, ZmHYD3 and ZmHYD4, have been functionally characterized thus far (Vallabhaneni et al, 2009;Yan et al, 2010).…”

“…In potato tubers, a polymorphism at a β-hydroxylase locus was shown to at least partially account for variation in β-carotene accumulation (Brown et al, 2006). In maize, ZmHYD3 displayed expression patterns that correlated with hydroxylated β-carotene derivative (β-xanthophyll) accumulation during endosperm development (Vallabhaneni et al, 2009). In addition, polymorphisms within the β-hydroxylase gene (CrtR-B1) are associated with a major QTL that controls β-carotene content in maize grains (Yan et al, 2010).…”