Dietary vitamin A deficiency causes eye disease in 40 million children each year and places 140 to 250 million at risk for health disorders. Many children in sub-Saharan Africa subsist on maize-based diets. Maize displays considerable natural variation for carotenoid composition, including vitamin A precursors alpha-carotene, beta-carotene, and beta-cryptoxanthin. Through association analysis, linkage mapping, expression analysis, and mutagenesis, we show that variation at the lycopene epsilon cyclase (lcyE) locus alters flux down alpha-carotene versus beta-carotene branches of the carotenoid pathway. Four natural lcyE polymorphisms explained 58% of the variation in these two branches and a threefold difference in provitamin A compounds. Selection of favorable lcyE alleles with inexpensive molecular markers will now enable developing-country breeders to more effectively produce maize grain with higher provitamin A levels.

Metabolic engineering of plant carotenoids in food crops has been a recent focus for improving human health. Pathway manipulation is predicated on comprehensive knowledge of this biosynthetic pathway, which has been extensively studied. However, there existed the possibility of an additional biosynthetic step thought to be dispensable because it could be compensated for by light. This step, mediated by a putative Z-ISO, was predicted to occur in the sequence of redox reactions that are coupled to an electron transport chain and convert the colorless 15-cis-phytoene to the red-colored all-trans-lycopene. The enigma of carotenogenesis in the absence of light (e.g. in endosperm, a target for improving nutritional content) argued for Z-ISO as a pathway requirement. Therefore, understanding of plant carotenoid biosynthesis was obviously incomplete. To prove the existence of Z-ISO, maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) mutants were isolated and the gene identified. Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9#-tri-cis-z-carotene, proving that Z-ISO encoded the missing step. Z-ISO was found to be important for both light-exposed and "dark" tissues. Comparative genomics illuminated the origin of Z-ISO found throughout higher and lower plants, algae, diatoms, and cyanobacteria. Z-ISO evolved from an ancestor related to the NnrU (for nitrite and nitric oxide reductase U ) gene required for bacterial denitrification, a pathway that produces nitrogen oxides as alternate electron acceptors for anaerobic growth. Therefore, plant carotenogenesis evolved by recruitment of genes from noncarotenogenic bacteria.

Abscisic acid (ABA) plays a vital role in mediating abiotic stress responses in plants. De novo ABA biosynthesis involves cleavage of carotenoid precursors by 9-cis-epoxycarotenoid dioxygenase (NCED), which is rate controlling in leaves and roots; however, additional bottlenecks in roots must be overcome, such as biosynthesis of upstream carotenoid precursors. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis; with PSY3 described here, maize (Zea mays) and other members of the Poaceae have three paralogous genes, in contrast to only one in Arabidopsis thaliana. PSY gene duplication has led to subfunctionalization, with each paralog exhibiting differential gene expression. We showed that PSY3 encodes a functional enzyme for which maize transcript levels are regulated in response to abiotic stresses, drought, salt, and ABA. Drought-stressed roots showed elevated PSY3 transcripts and ABA, responses reversed by rehydration. By blocking root carotenoid biosynthesis with the maize y9 mutation, we demonstrated that PSY3 mRNA elevation correlates with carotenoid accumulation and that blocking carotenoid biosynthesis interferes with stress-induced ABA accumulation. In parallel, we observed elevated NCED transcripts and showed that, in contrast to dicots, root zeaxanthin epoxidase transcripts were unchanged. PSY3 was the only paralog for which transcripts were induced in roots and abiotic stress also affected leaf PSY2 transcript levels; PSY1 mRNA was not elevated in any tissues tested. Our results suggest that PSY3 expression influences root carotenogenesis and defines a potential bottleneck upstream of NCED; further examination of PSY3 in the grasses is of value for better understanding root-specific stress responses that impact plant yield.

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Carotenoids are essential for photosynthesis and photoprotection; they also serve as precursors to signaling molecules that influence plant development and biotic/abiotic stress responses. With potential to improve plant yield and nutritional quality, carotenoids are targets for metabolic breeding/engineering, particularly in the Poaceae (grass family), which includes the major food crops. Depending on genetic background, maize (Zea mays) endosperm carotenoid content varies, and therefore breeding-enhanced carotenoid levels have been of ongoing interest. The first committed step in the plastid-localized biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). The gene family in maize and other grasses contains three paralogs with specialized roles that are not well understood. Maize endosperm carotenoid accumulation requires PSY1 expression. A maize antibody was used to localize PSY1 to amyloplast envelope membranes and to determine PSY1 accumulation in relation to carotenoid accumulation in developing endosperm. To test when and if PSY transcript levels correlated with carotenoid content, advantage was taken of a maize germplasm diversity collection that exhibits genetic and chemical diversity. Total carotenoid content showed statistically significant correlation with endosperm transcript levels at 20 d after pollination for PSY1 but not PSY2 or PSY3. Timing of PSY1 transcript abundance, previously unknown, provides critical information for choosing breeding alleles or properly controlling introduced transgenes. PSY1 was unexpectedly found to have an additional role in photosynthetic tissue, where it was required for carotenogenesis in the dark and for heat stress tolerance. Leaf carotenogenesis was shown to require phytochrome-dependent and phytochrome-independent photoregulation of PSY2 plus nonphotoregulated PSY1 expression.

Despite ongoing research on carotenoid biosynthesis in model organisms, there is a paucity of information on pathway regulation operating in the grasses (Poaceae), which include plants of world-wide agronomic importance. As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results. In comparison to maize (Zea mays), rice (Oryza sativa) accumulates no endosperm carotenoids, despite having a functional pathway in chloroplasts. To better understand why these two related grasses differ in endosperm carotenoid content, we began to characterize genes encoding phytoene synthase (PSY), since this nuclear-encoded enzyme appeared to catalyze a rate-controlling step in the plastid-localized biosynthetic pathway. The enzyme had been previously associated with the maize Y1 locus thought to be the only functional gene controlling PSY accumulation, though function of the Y1 gene product had never been demonstrated. We show that both maize and rice possess and express products from duplicate PSY genes, PSY1 (Y1) and PSY2; PSY1 transcript accumulation correlates with carotenoid-containing endosperm. Using a heterologous bacterial system, we demonstrate enzyme function of PSY1 and PSY2 that are largely conserved in sequence except for N-and C-terminal domains. By database mining and use of ortholog-specific universal PCR primers, we found that the PSY duplication is prevalent in at least eight subfamilies of the Poaceae, suggesting that this duplication event preceded evolution of the Poaceae. These findings will impact study of grass phylogeny and breeding of enhanced carotenoid content in an entire taxonomic group of plant crops critical for global food security.Carotenoids, a class of over 600 structures derived from isoprenoids, are synthesized by all photosynthetic organisms, some bacteria, and fungi. In plants, carotenoids are essential for plant growth and development; mutations blocking carotenoid accumulation have pleiotropic effects on chloroplast biogenesis and seed development (Robertson et al., 1978;Wurtzel, 1992). Carotenoids function as accessory pigments in photosynthesis, as photoprotectors preventing photooxidative damage, and as precursors to the plant hormone, abscisic acid (Hirschberg, 2001). The presence of carotenoids in plant endosperm tissue adds nutritional value; in humans and animals, dietary carotenoids are essential precursors to vitamin A and to retinoid compounds needed in development (Lee et al., 1981;Bendich and Olson, 1989). Nonprovitamin A carotenoids, such as lycopene, lutein, zeaxanthin, and others, also play beneficial roles in human health (Giovannucci et al., 1995;Kohlmeier et al., 1997;Sommerburg et al., 1998;Krinsky et al., 2003). The various roles of carotenoids affecting plant yield and nutritional potential has made them targets for breeding and metabolic engineering (Shewmaker et al., 1999;Matthews and Wurtzel, 2000;Ye et al., 2000;Davison, 2002;Blott et al., 2003;Gallagher et al., ...

Carotenoids are a diverse group of pigments found in plants, fungi, and bacteria. They serve essential functions in plants and provide health benefits for humans and animals. In plants, it was thought that conversion of the C40 carotenoid backbone, 15-cisphytoene, to all-trans-lycopene, the geometrical isomer required by downstream enzymes, required two desaturases (phytoene desaturase and z-carotene desaturase [ZDS]) plus a carotene isomerase (CRTISO), in addition to light-mediated photoisomerization of the 15-cis-double bond; bacteria employ only a single enzyme, CRTI. Characterization of the maize (Zea mays) pale yellow9 (y9) locus has brought to light a new isomerase required in plant carotenoid biosynthesis. We report that maize Y9 encodes a factor required for isomerase activity upstream of CRTISO, which we term Z-ISO, an activity that catalyzes the cis-to trans-conversion of the 15-cis-bond in 9,15,9#-tri-cis-z-carotene, the product of phytoene desaturase, to form 9,9#-di-cis-z-carotene, the substrate of ZDS. We show that recessive y9 alleles condition accumulation of 9,15,9#-tri-cis-z-carotene in dark tissues, such as roots and etiolated leaves, in contrast to accumulation of 9,9#-di-cis-z-carotene in a ZDS mutant, viviparous9. We also identify a locus in Euglena gracilis, which is similarly required for Z-ISO activity. These data, taken together with the geometrical isomer substrate requirement of ZDS in evolutionarily distant plants, suggest that Z-ISO activity is not unique to maize, but will be found in all higher plants. Further analysis of this new gene-controlled step is critical to understanding regulation of this essential biosynthetic pathway.

Enhancement of the carotenoid biosynthetic pathway in food crops benefits human health and adds commercial value of natural food colorants. However, predictable metabolic engineering or breeding is limited by the incomplete understanding of endogenous pathway regulation, including rate-controlling steps and timing of expression in carotenogenic tissues. The grass family (Poaceae) contains major crop staples, including maize (Zea mays), wheat (Triticum aestivum), rice (Oryza sativa), sorghum (Sorghum bicolor), and millet (Pennisetum glaucum). Maize carotenogenesis was investigated using a novel approach to discover genes encoding limiting biosynthetic steps in the nutritionally targeted seed endosperm. A combination of bioinformatics and cloning were first used to identify and map gene families encoding enzymes in maize and other grasses. These enzymes represented upstream pathways for isopentenyl diphosphate and geranylgeranyl diphosphate synthesis and the downstream carotenoid biosynthetic pathway, including conversion to abscisic acid. A maize germplasm collection was used for statistical testing of the correlation between carotenoid content and candidate gene transcript levels. Multiple pathway bottlenecks for isoprenoid biosynthesis and carotenoid biosynthesis were discovered in specific temporal windows of endosperm development. Transcript levels of paralogs encoding isoprenoid isopentenyl diphosphate and geranylgeranyl diphosphate-producing enzymes, DXS3, DXR, HDR, and GGPPS1, were found to positively correlate with endosperm carotenoid content. For carotenoid pathway enzymes, transcript levels for CrtISO inversely correlated with seed carotenoid content, as compared with positive correlation of PSY1 transcripts. Since zeaxanthin epoxidase (ZEP) depletes the carotenoid pool in subsequent conversion to abscisic acid, ZEP transcripts were examined. Carotenoid accumulation was found to be inversely associated with ZEP1 and ZEP2 transcript levels. Extension of the maize results using phylogenetic analysis identified orthologs in other grass species that may serve as potential metabolic engineering targets.