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.

ZmPBF and ZmGAMYB transcription factors independently transactivate the promoter of the maize (Zea mays) ß-carotene hydroxylase 2 gene.

The maize (Zea mays) enzyme ß-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of ß-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036 bp of the 5'-flanking region containing the 263 bp 5'-untranslated region (5'-UTR) including the first intron. We linked this to the ß-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5'-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.

Prevalence of individual and bulk tank milk antibodies of bovine herpesvirus type 1 and its relation to milk quality parameters on dairy farms in Catalonia (north-east Spain).

Bovine herpesvirus type 1 (BoHV-1) is the causative agent for infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cows or balanoposthitis in bulls. In this study, individual and bulk tank milk (BTM) samples from 5 Catalan dairy farms with different control strategies against BoHV-1 were analysed during the course of a year for milk quality parameters and glycoprotein E (gE) antibodies. Detection of gE antibodies was carried out with ELISA techniques. Prevalence of BoHV-1 varied between farms, and was stable during the study in individual and BTM samples. Comparing the antibody results of samples with milk quality parameters, positive samples with higher levels of antibodies corresponded to lower lactose and to higher percentages of fat and somatic cells.

Provitamin A carotenoids from an engineered high-carotenoid maize are bioavailable and zeaxanthin does not compromise ß-carotene absorption in poultry.

High-carotenoid (HC) maize, a biofortified staple crop which accumulates ß-carotene, ß-cryptoxanthin, lutein and zeaxanthin, was used as a feed component in a chicken feeding trial to assess the bioavailability of provitamin A (PVA) carotenoids in the kernel matrix compared to the synthetic and natural color additives routinely used in the poultry industry. We found that the PVA carotenoids in HC maize were not metabolized in the same manner: ß-carotene was preferentially converted into retinol in the intestine whereas ß-cryptoxanthin accumulated in the liver. We also considered the effect of zeaxanthin on the absorption of PVA carotenoids because zeaxanthin is the major carotenoid component of HC maize. We found that chickens fed on diets with low levels of zeaxanthin accumulated higher levels of retinol in the liver, suggesting that zeaxanthin might interfere with the absorption of ß-carotene, although this observation was not statistically significant. Our results show that HC maize provides bioavailable carotenoids, including PVA carotenoids, and is suitable for use as a feed component.

The distribution of carotenoids in hens fed on biofortified maize is influenced by feed composition, absorption, resource allocation and storage.

Carotenoids are important dietary nutrients with health-promoting effects. The biofortification of staple foods with carotenoids provides an efficient delivery strategy but little is known about the fate and distribution of carotenoids supplied in this manner. The chicken provides a good model of human carotenoid metabolism so we supplemented the diets of laying hens using two biofortified maize varieties with distinct carotenoid profiles and compared the fate of the different carotenoids in terms of distribution in the feed, the hen's livers and the eggs. We found that after a period of depletion, pro-vitamin A (PVA) carotenoids were preferentially diverted to the liver and relatively depleted in the eggs, whereas other carotenoids were transported to the eggs even when the liver remained depleted. When retinol was included in the diet, it accumulated more in the eggs than the livers, whereas PVA carotenoids showed the opposite profile. Our data suggest that a transport nexus from the intestinal lumen to the eggs introduces bottlenecks that cause chemically-distinct classes of carotenoids to be partitioned in different ways. This nexus model will allow us to optimize animal feed and human diets to ensure that the health benefits of carotenoids are delivered in the most effective manner.

A WUR SNP is associated with European Porcine Reproductive and Respiratory Virus Syndrome resistance and growth performance in pigs.

Porcine reproductive and respiratory syndrome (PRRS) causes decreased reproductive performance and respiratory problems in pigs. The goals of the current study were 1) to examine whether individual variation applies to infection with PRRSV European strains and 2) to investigate the association of a single nucleotide polymorphism (SNP) WUR10000125 (WUR) at the interferon-inducible guanylate-binding protein 1 gene (GBP1) with average daily gain (ADG) in PRRSV infected and uninfected pigs. The experimental procedure consisted of two trials in which pigs from negative PRRSV farms were infected with a wild-type (n=80) or vaccinated with an attenuated European PRRS virus strain (n=40) and then monitored after infection or vaccination. Viral load and ADG were determined for each pig. In a third trial, the ADG for PRRSV-free pigs was monitored. All pigs were genotyped for WUR at the GBP1 gene (AA and AG genotype were defined). Results indicated that there was individual variation in the viral load from pigs challenged with a wild-type or low virulent European PRRSV strain. Secondly, our data showed that WUR SNP was associated to ADG in vaccinated pigs. Thus, ADG in AG pigs was significantly higher than in AA ones after vaccinating with an attenuated PRRSV strain. However, the reverse happened in a PRRSV-free environment where the AA pigs were those that grew faster. Based on these results, there is a scope for selecting pigs according to their responses to PRRS virus infection with European strains and that WUR SNP may play a role in causing PRRSV resistance.

Carotenoid-enriched transgenic corn delivers bioavailable carotenoids to poultry and protects them against coccidiosis.

Carotenoids are health-promoting organic molecules that act as antioxidants and essential nutrients. We show that chickens raised on a diet enriched with an engineered corn variety containing very high levels of four key carotenoids (ß-carotene, lycopene, zeaxanthin and lutein) are healthy and accumulate more bioavailable carotenoids in peripheral tissues, muscle, skin and fat, and more retinol in the liver, than birds fed on standard corn diets (including commercial corn supplemented with colour additives). Birds were challenged with the protozoan parasite Eimeria tenella and those on the high-carotenoid diet grew normally, suffered only mild disease symptoms (diarrhoea, footpad dermatitis and digital ulcers) and had lower faecal oocyst counts than birds on the control diet. Our results demonstrate that carotenoid-rich corn maintains poultry health and increases the nutritional value of poultry products without the use of feed additives.