Vitamin A deficiency is a global health burden. This deficiency can be alleviated through provitamin A carotenoid biofortification of maize and other Poaceae crops. However, the predictive metabolic engineering or breeding is limited by the incomplete understanding of endogenous pathway regulation. If the pathway regulation was better understood, enhancement of carotenoid biosynthesis could be controlled by limiting rate-controlling steps and timing of expression in carotenogeneic tissues.

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 transcript profiling were first used to identify, map and study expression analysis of gene families encoding enzymes in maize and other grasses. These enzymes include the methylerythritol 4-phosphate MEP pathway for synthesis of IPP and GGPP, the downstream carotenoid biosynthetic pathway and as well as those involved in degradation of carotenoids.

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. This core collection was used for statistical testing of correlation between carotenoid content and candidate gene transcript levels. Multiple pathway bottlenecks for isoprenoid biosynthesis and carotenoid biosynthesis controlling both total carotenoid content and individual carotenoids were discovered in specific temporal windows of endosperm development. Transcript levels of paralogs encoding isoprenoid IPP and GGPP-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 to positive correlation of PSY1 transcripts. Among genes encoding enzymes controlling individual carotenoids, LCYE was shown to control the ratio of two branches of the pathway, and HYD3 was shown to be associated in converting provitamin A compounds to non-provitmain A compounds. Three natural alleles of HYD3 in 51 maize lines explained 78% of variation and ∼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.

Further downstream, since ZEP depletes the carotenoid pool in subsequent conversion to ABA, ZEP transcripts were examined. Carotenoid accumulation was found to be inversely associated with ZEP1 and ZEP2 transcript levels. Additionally, degradation of carotenoids is another mechanism to reduce the level of carotenoids in any tissue. Degradation of carotenoids is controlled by a small gene family of eleven carotenoid cleavage genes. Although there is no direct evidence between mRNA levels of these genes and total carotenoid content, preliminary analysis suggested that copy number may be associated with reduced endosperm carotenoids.

Degradation of ABA by the action of ABA hydroxylases was also studied. The enzymatic activity of ABA 8’-hydroxylase is considered as one of the key steps in maintaining ABA levels necessary for physiological processes. ABA8Ox genes were identified in maize and respective gene expression was found in all tissues, with a high degree of specificity to each tissue and some degree of overlap. ZmABA8Ox1a and ZmABA8Ox1b were shown to be the major transcript components for regulating ABA catabolism in drought-stressed roots.

Extension of the maize results using phylogenetic analysis identified orthologs in other Grass species that may serve as potential metabolic engineering targets.