Multilevel interactions between native and ectopic isoprenoid pathways affect global metabolism in rice.

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.

Engineered Maize Hybrids with Diverse Carotenoid Profiles and Potential Applications in Animal Feeding.

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a "mutant series," allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of ß-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high ß:ε ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low ß:ε ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a ß-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic ß-carotene ketolase with a superior ß-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

Timing of Pulmonary Rehabilitation in Readmitted Patients with Severe Chronic Obstructive Pulmonary Disease: A Randomized Clinical Trial.

Early pulmonary rehabilitation (PR), started during hospitalization or within the first month after discharge, has been shown to reduce exacerbations and improve health-related-quality of life (HRQoL) and exercise capacity. However, no randomized clinical trials (RCT) have compared the efficacy of PR started during hospitalization (DHPR) to PR initiated one month post-hospitalization (PHPR). We conducted an RCT to compare DHPR to PHPR in severe patients with COPD readmitted for exacerbations in a tertiary hospital setting. Patients were randomized to receive three months of DHPR or PHPR. Outcomes were assessed at completion of the PR programme and at months 3 and 9. A total of 53 patients (26 DHPR and 27 PHPR) were included. There were no between-group differences in the number of exacerbations (mean, 3.62 vs. 3.04 in the DHPR and PHPR groups, respectively; p = 0.403). Dyspnea in activities of daily living, exercise capacity, and all HRQoL parameters improved in the PHPR group. In the DHPR group, improvement was observed only for some HRQoL parameters. All gains in both groups were lost during follow-up. More adverse events were observed in the DHPR group (20 vs 5, p = 0.023), although none of these were clinically significant. In this sample of patients with severe COPD readmitted to the hospital for exacerbations, both approaches to PR were safe, but PHPR yielded better outcomes overall. These findings suggest that, PR should be initiated in patients with severe COPD only after hospital discharge when the patients' clinical condition has stabilized.

CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.

KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

Unexpected synergistic HIV neutralization by a triple microbicide produced in rice endosperm.

The transmission of HIV can be prevented by the application of neutralizing monoclonal antibodies and lectins. Traditional recombinant protein manufacturing platforms lack sufficient capacity and are too expensive for developing countries, which suffer the greatest disease burden. Plants offer an inexpensive and scalable alternative manufacturing platform that can produce multiple components in a single plant, which is important because multiple components are required to avoid the rapid emergence of HIV-1 strains resistant to single microbicides. Furthermore, crude extracts can be used directly for prophylaxis to avoid the massive costs of downstream processing and purification. We investigated whether rice could simultaneously produce three functional HIV-neutralizing proteins (the monoclonal antibody 2G12, and the lectins griffithsin and cyanovirin-N). Preliminary in vitro tests showed that the cocktail of three proteins bound to gp120 and achieved HIV-1 neutralization. Remarkably, when we mixed the components with crude extracts of wild-type rice endosperm, we observed enhanced binding to gp120 in vitro and synergistic neutralization when all three components were present. Extracts of transgenic plants expressing all three proteins also showed enhanced in vitro binding to gp120 and synergistic HIV-1 neutralization. Fractionation of the rice extracts suggested that the enhanced gp120 binding was dependent on rice proteins, primarily the globulin fraction. Therefore, the production of HIV-1 microbicides in rice may not only reduce costs compared to traditional platforms but may also provide functional benefits in terms of microbicidal potency.

CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the corresponding small subunit gene APS2b in rice leaves.

The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.

The Arabidopsis ORANGE (AtOR) gene promotes carotenoid accumulation in transgenic corn hybrids derived from parental lines with limited carotenoid pools.

KEY MESSAGE: The AtOR gene enhances carotenoid levels in corn by promoting the formation of plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant. The cauliflower orange (or) gene mutation influences carotenoid accumulation in plants by promoting the transition of proplastids into chromoplasts, thus creating intracellular storage compartments that act as metabolic sink. We overexpressed the Arabidopsis OR gene under the control of the endosperm-specific wheat LMW glutenin promoter in a white corn variety that normally accumulates only trace amounts of carotenoids. The total endosperm carotenoid content in the best-performing AtOR transgenic corn line was 32-fold higher than wild-type controls (~25 µg/g DW at 30 days after pollination) but the principal carotenoids remained the same, suggesting that AtOR increases the abundance of existing carotenoids without changing the metabolic composition. We analyzed the expression of endogenous genes representing the carotenoid biosynthesis and MEP pathways, as well as the plastid fusion/translocation factor required for chromoplast formation, but only the DXS1 gene was upregulated in the transgenic corn plants. The line expressing AtOR at the highest level was crossed with four transgenic corn lines expressing different carotenogenic genes and accumulating different carotenoids. The introgression of AtOR increased the carotenoid content of the hybrids when there was a limited carotenoid pool in the parental line, but had no effect when carotenoids were already abundant in the parent. The AtOR gene therefore appears to enhance carotenoid levels by promoting the formation of carotenoid-sequestering plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant because high levels of carotenoids can induce the formation of carotenoid-sequestering plastoglobuli even in the absence of AtOR.

Biofortification of crops with nutrients: factors affecting utilization and storage.

Biofortification is an effective and economical method to improve the micronutrient content of crops, particularly staples that sustain human populations in developing countries. Whereas conventional fortification requires artificial additives, biofortification involves the synthesis or accumulation of nutrients by plants at source. Little is known about the relative merits of biofortification and artificial fortification in terms of nutrient bioaccessibility and bioavailability, and much depends on the biochemical nature of the nutrient, which can promote or delay uptake, and determine how efficiently different nutrients are transported through the blood, stored, and utilized. Data from the first plants biofortified with minerals and vitamins provide evidence that the way in which nutrients are presented can affect how they are processed and utilized in the human body. The latest studies on the effects of the food matrix, processing and storage on nutrient transfer from biofortified crops are reviewed, as well as current knowledge about nutrient absorption and utilization.

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous ß-carotene hydroxylase activity.

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from ß-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by ß-carotene hydroxylase and ß-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii ß-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

Red Anthocyanins and Yellow Carotenoids Form the Color of Orange-Flower Gentian (Gentiana lutea L. var. aurantiaca).

Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.

Patterns of CRISPR/Cas9 activity in plants, animals and microbes.

The CRISPR/Cas9 system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on-target mutations), the targeting accuracy (likelihood of off-target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome-editing tools.

Engineered maize as a source of astaxanthin: processing and application as fish feed.

Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90 % of the oil. The resulting astaxanthin raw material contained non-esterified astaxanthin with 12 % 4-keto zeaxanthin and 2 % zeaxanthin as additional carotenoids. Isomeric analysis confirmed the exclusive presence of the 3S, 3'S astaxanthin enantiomer. The geometrical isomers were 89 % all-E, 8 % 13-Z and 3 % 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 7 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 µg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.

Identification of line-specific strategies for improving carotenoid production in synthetic maize through data-driven mathematical modeling.

Plant synthetic biology is still in its infancy. However, synthetic biology approaches have been used to manipulate and improve the nutritional and health value of staple food crops such as rice, potato and maize. With current technologies, production yields of the synthetic nutrients are a result of trial and error, and systematic rational strategies to optimize those yields are still lacking. Here, we present a workflow that combines gene expression and quantitative metabolomics with mathematical modeling to identify strategies for increasing production yields of nutritionally important carotenoids in the seed endosperm synthesized through alternative biosynthetic pathways in synthetic lines of white maize, which is normally devoid of carotenoids. Quantitative metabolomics and gene expression data are used to create and fit parameters of mathematical models that are specific to four independent maize lines. Sensitivity analysis and simulation of each model is used to predict which gene activities should be further engineered in order to increase production yields for carotenoid accumulation in each line. Some of these predictions (e.g. increasing Zmlycb/Gllycb will increase accumulated ß-carotenes) are valid across the four maize lines and consistent with experimental observations in other systems. Other predictions are line specific. The workflow is adaptable to any other biological system for which appropriate quantitative information is available. Furthermore, we validate some of the predictions using experimental data from additional synthetic maize lines for which no models were developed.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid.

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a ß-carotene hydroxylase and a ß-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the ß-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Knowledge-driven approaches for engineering complex metabolic pathways in plants.

Plant metabolic pathways are complex and often feature multiple levels of regulation. Until recently, metabolic engineering in plants relied on the laborious testing of ad hoc modifications to achieve desirable changes in the metabolic profile. However, technological advances in data mining, modeling, multigene engineering and genome editing are now taking away much of the guesswork by allowing the impact of modifications to be predicted more accurately. In this review we discuss recent developments in knowledge-based metabolic engineering strategies, that is the gathering and mining of genomic, transcriptomic, proteomic and metabolomic data to generate models of metabolic pathways that help to define and refine optimal intervention strategies.

Engineering complex metabolic pathways in plants.

Metabolic engineering can be used to modulate endogenous metabolic pathways in plants or introduce new metabolic capabilities in order to increase the production of a desirable compound or reduce the accumulation of an undesirable one. In practice, there are several major challenges that need to be overcome, such as gaining enough knowledge about the endogenous pathways to understand the best intervention points, identifying and sourcing the most suitable metabolic genes, expressing those genes in such a way as to produce a functional enzyme in a heterologous background, and, finally, achieving the accumulation of target compounds without harming the host plant. This article discusses the strategies that have been developed to engineer complex metabolic pathways in plants, focusing on recent technological developments that allow the most significant bottlenecks to be overcome.

Engineering metabolic pathways in plants by multigene transformation.

Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.

A question of balance: achieving appropriate nutrient levels in biofortified staple crops.

The biofortification of staple crops with vitamins is an attractive strategy to increase the nutritional quality of human food, particularly in areas where the population subsists on a cereal-based diet. Unlike other approaches, biofortification is sustainable and does not require anything more than a standard food-distribution infrastructure. The health-promoting effects of vitamins depend on overall intake and bioavailability, the latter influenced by food processing, absorption efficiency and the utilisation or retention of the vitamin in the body. The bioavailability of vitamins in nutritionally enriched foods should ideally be adjusted to achieve the dietary reference intake in a reasonable portion. Current vitamin biofortification programmes focus on the fat-soluble vitamins A and E, and the water-soluble vitamins C and B9 (folate), but the control of dosage and bioavailability has been largely overlooked. In the present review, we discuss the vitamin content of nutritionally enhanced foods developed by conventional breeding and genetic engineering, focusing on dosage and bioavailability. Although the biofortification of staple crops could potentially address micronutrient deficiency on a global scale, further research is required to develop effective strategies that match the bioavailability of vitamins to the requirements of the human diet.

Ascorbic acid synthesis and metabolism in maize are subject to complex and genotype-dependent feedback regulation during endosperm development.

L-ascorbic acid (vitamin C) is an antioxidant and electron donor whose metabolism in plants is under strict feedback control. The factors that influence L-ascorbic acid accumulation in staple crops are only partially understood. One way to gain insight into the regulation of L-ascorbic acid metabolism is to investigate the endogenous pathways in various genetic backgrounds and characterize their interactions with transgenes encoding relevant enzymes. In an initial step, we investigated the developmental profile of L-ascorbic acid accumulation in the endosperm of three diverse maize genotypes and a transgenic line expressing rice dehydroascorbate reductase, which enhances L-ascorbic acid recycling. We determined the transcript levels of all the key genes in the L-ascorbic acid metabolic pathways as well as the specific levels of ascorbic acid and dehydroascorbate. L-ascorbic acid levels were high 20 days after pollination and declined thereafter. We found significant genotype-dependent variations in the transcript levels of some genes, with particular complexity in the ascorbic acid recycling pathway. Our data will help to elucidate the complex mechanisms underlying the regulation of L-ascorbic acid metabolism in plants, particularly the impact of genetic background on the strict regulation of ascorbic acid metabolism in endosperm cells.