Maize provitamin a carotenoids, current resources, and future metabolic engineering challenges.

Vitamin A deficiency is a serious global health problem that can be alleviated by improved nutrition. Development of cereal crops with increased provitamin A carotenoids can provide a sustainable solution to eliminating vitamin A deficiency worldwide. Maize is a model for cereals and a major staple carbohydrate source. Here, we discuss maize carotenogenesis with regard to pathway regulation, available resources, and current knowledge for improving carotenoid content and levels of provitamin A carotenoids in edible maize endosperm. This knowledge will be applied to improve the nutritional composition of related Poaceae crops. We discuss opportunities and challenges for optimizing provitamin A carotenoid biofortification of cereal food crops.

A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana.

BACKGROUND: The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana. RESULTS: A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of chlorophyll biosynthesis genes in a manner that is consistent with the increased synthesis of carotenoid precursors for ABA biosynthesis. In all tissues examined, induction of ß-carotene hydroxylase transcript levels are linked to an increased demand for ABA. CONCLUSIONS: This analysis provides compelling evidence to suggest that coordinated transcriptional regulation of isoprenoid-related biosynthesis pathway genes plays a major role in coordinating the synthesis of functionally related chloroplast localized isoprenoid-derived compounds.

The carotenoid dioxygenase gene family in maize, sorghum, and rice.

Carotenoids and their apocarotenoid derivatives play essential physiological and developmental roles and provide plants tolerance to a variety of stresses. Carotenoid cleavage dioxygenases mediate the degradation of carotenoids to apocarotenoids. A better understanding of biosynthesis vs. degradation could be useful for controlling carotenoid levels leading to improved plant fitness and/or enhanced content of nutritionally valuable carotenoids. The Poaceae (grass) plant family contains many crops of agronomic value. Therefore this study focused on characterizing the carotenoid dioxygenase gene family in the grass species maize, rice, and sorghum with comparison made to newly identified gene families in two non-seed plants as well as an alga and previously identified eudicot genes. Genome analysis was used to map grass genes encoding the carotenoid dioxygenases to chromosome locations. Sequences of encoded proteins were phylogenetically compared. CCD8b was identified as a new class of cleavage dioxygenases that may play a specialized role in apocarotenoid biogenesis. A simple PCR assay was developed to measure CCD1 gene copy number which is known to vary in maize. Using a panel of maize inbred lines varying in carotenoid content, linear regression analysis revealed a statistically significant negative correlation between copy number of CCD1 and carotenoid content, an effect likely mediated through the resulting elevated levels of endosperm CCD1 transcripts in high copy number lines. The PCR assay adds to a growing toolbox for metabolic engineering of maize endosperm carotenoids. This new tool can be used to select maize lines that are less likely to promote endosperm carotenoid degradation, thus predicting optimal results in metabolic engineering of endosperm provitamin A and/or nonprovitamin A carotenoids.

From epoxycarotenoids to ABA: the role of ABA 8'-hydroxylases in drought-stressed maize roots.

The ability of plants to withstand drought, a potentially major constraint to yield and production, is influenced by abscisic acid (ABA). ABA is synthesized in the cytosol from plastid carotenoid pathway derived precursors, and later inactivated by the action of ABA hydroxylases. Endogenous accumulation of ABA is controlled by both its synthesis and catabolism. Enzymatic activity of ABA 8'-hydroxylase (ABA8Ox), also referred to as CYP707A, is considered one of the key steps in modulating ABA levels that control numerous physiological processes. To investigate the role of this enzyme, maize ABA8Ox gene family members were identified. ABA8Ox gene expression was then analyzed in different tissues and roots during the drought-stress response in maize. These genes were found to be expressed in all tissues, with a high degree of specificity to each tissue and some degree of overlap. Maize ABA8Ox1a and ABA8Ox1b were shown to be the major transcript components for regulating ABA catabolism in drought-stressed roots. Phylogenetic and gene-structure analyses were performed to extend the implications and infer the cause of ABA catabolism in other cereal crops.

Metabolite sorting of a germplasm collection reveals the hydroxylase3 locus as a new target for maize provitamin A biofortification.

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 beta-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 beta-carotene relative to beta-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of beta-carotene. A simple PCR assay to track and identify Hydroxylase3 alleles will be valuable for predicting nutritional content in genetically diverse cultivars found worldwide.

Timing and biosynthetic potential for carotenoid accumulation in genetically diverse germplasm of maize.

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.

The maize phytoene synthase gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and thermal stress tolerance.

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.

Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification.

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.

PSY3, a new member of the phytoene synthase gene family conserved in the Poaceae and regulator of abiotic stress-induced root carotenogenesis.

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.