Carotenoids moderate the effectiveness of a Bt gene against the European corn borer, Ostrinia nubilalis.

We assessed the effectiveness of a biofortified maize line (4BtxHC) which accumulates high levels of antioxidant carotenoids that also expressed the insecticidal Cry1Ac Bacillus thuringiensis (Bt) gene against the European corn borer Ostrinia nubilalis. This line had been previously engineered to accumulate carotenoids specifically in the seed endosperm, whereas the Bt gene was expressed constitutively. The concentrations of Bt toxin (Cry 1Ac) in the leaves of the 4Bt and 4BtxHC lines were not significantly different at 47±6 µg/g of fresh weight (FW); neither were they in the kernels of both lines (35±3 µg/g FW). The kernels and leaves were toxic to the larvae of O. nubilalis. However, the insecticidal activity was substantially lower (ca. 20%) than that of lines that expressed only Bt in spite that the two lines showed a quantity of toxin not significantly different in kernels or in leaves. Although the reduced effectiveness of Cry1Ac in kernels may not be entirely surprising, the observation of the same phenomenon in vegetative tissues was unexpected. When semi-artificial diets containing kernels from 4Bt supplemented with different levels of ß-carotene were used in insect bioassays, the ß-carotene moderated the effectiveness of the Bt similarly to the plant material with carotenoid enrichment. To elucidate the biochemical basis of the reduced effectiveness of Bt toxin in the carotenoid-enriched plants, we measured the activity of three enzymes known to be implicated in the detoxification defence, namely, catalase, superoxide dismutase and glutathione S-transferase. Whereas Cry1Ac expression significantly increased SOD and CAT enzymatic activity in the absence of carotenoids, carotenoids, either in 4BtxHC or in artificial diets enriched with ß-carotene, significantly lowered CAT activity. Carotenoids can therefore moderate the susceptibility of the maize borer O. nubilalis to Cry1Ac, and we hypothesize that their role as antioxidants could explain this phenomenon via their scavenging of reactive oxygen species produced during Cry1Ac detoxification in the larvae. The involvement of this mechanism in the decreased mortality caused by Cry1Ac when carotenoids are present in the diet is discussed.

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.

Can the world afford to ignore biotechnology solutions that address food insecurity?

Genetically engineered (GE) crops can be used as part of a combined strategy to address food insecurity, which is defined as a lack of sustainable access to safe and nutritious food. In this article, we discuss the causes and consequences of food insecurity in the developing world, and the indirect economic impact on industrialized countries. We dissect the healthcare costs and lost productivity caused by food insecurity, and evaluate the relative merits of different intervention programs including supplementation, fortification and the deployment of GE crops with higher yields and enhanced nutritional properties. We provide clear evidence for the numerous potential benefits of GE crops, particularly for small-scale and subsistence farmers. GE crops with enhanced yields and nutritional properties constitute a vital component of any comprehensive strategy to tackle poverty, hunger and malnutrition in developing countries and thus reduce the global negative economic effects of food insecurity.

The contribution of transgenic plants to better health through improved nutrition: opportunities and constraints.

Malnutrition is a prevalent and entrenched global socioeconomic challenge that reflects the combined impact of poverty, poor access to food, inefficient food distribution infrastructure, and an over-reliance on subsistence mono-agriculture. The dependence on staple cereals lacking many essential nutrients means that malnutrition is endemic in developing countries. Most individuals lack diverse diets and are therefore exposed to nutrient deficiencies. Plant biotechnology could play a major role in combating malnutrition through the engineering of nutritionally enhanced crops. In this article, we discuss different approaches that can enhance the nutritional content of staple crops by genetic engineering (GE) as well as the functionality and safety assessments required before nutritionally enhanced GE crops can be deployed in the field. We also consider major constraints that hinder the adoption of GE technology at different levels and suggest policies that could be adopted to accelerate the deployment of nutritionally enhanced GE crops within a multicomponent strategy to combat malnutrition.

Biofortification of plants with altered antioxidant content and composition: genetic engineering strategies.

Antioxidants are protective molecules that neutralize reactive oxygen species and prevent oxidative damage to cellular components such as membranes, proteins and nucleic acids, therefore reducing the rate of cell death and hence the effects of ageing and ageing-related diseases. The fortification of food with antioxidants represents an overlap between two diverse environments, namely fortification of staple foods with essential nutrients that happen to have antioxidant properties (e.g. vitamins C and E) and the fortification of luxury foods with health-promoting but non-essential antioxidants such as flavonoids as part of the nutraceuticals/functional foods industry. Although processed foods can be artificially fortified with vitamins, minerals and nutraceuticals, a more sustainable approach is to introduce the traits for such health-promoting compounds at source, an approach known as biofortification. Regardless of the target compound, the same challenges arise when considering the biofortification of plants with antioxidants, that is the need to modulate endogenous metabolic pathways to increase the production of specific antioxidants without affecting plant growth and development and without collateral effects on other metabolic pathways. These challenges become even more intricate as we move from the engineering of individual pathways to several pathways simultaneously. In this review, we consider the state of the art in antioxidant biofortification and discuss the challenges that remain to be overcome in the development of nutritionally complete and health-promoting functional foods.

Combinatorial genetic transformation of cereals and the creation of metabolic libraries for the carotenoid pathway.

Combinatorial nuclear transformation is used to generate populations of transgenic plants containing random selections from a collection of input transgenes. This is a useful approach because it provides the means to test different combinations of genes without the need for separate transformation experiments, allowing the comprehensive analysis of metabolic pathways and other genetic systems requiring the coordinated expression of multiple genes. The principle of combinatorial nuclear transformation is demonstrated in this chapter through protocols developed in our laboratory that allow combinations of genes encoding enzymes in the carotenoid biosynthesis pathway to be introduced into rice and a white-endosperm variety of corn. These allow the accumulation of carotenoids to be screened initially by the colour of the endosperm, which ranges from white through various shades of yellow and orange depending on the types and quantities of carotenoids present. The protocols cover the preparation of DNA-coated metal particles, the transformation of corn and rice plants by particle bombardment, the regeneration of transgenic plants, the extraction of carotenoids from plant tissues, and their analysis by high-performance liquid chromatography.

Bacillus thuringiensis: a century of research, development and commercial applications.

Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary phase of its growth cycle. The spores contain crystals, predominantly comprising one or more Cry and/or Cyt proteins (also known as δ-endotoxins) that have potent and specific insecticidal activity. Different strains of Bt produce different types of toxin, each of which affects a narrow taxonomic group of insects. Therefore, Bt toxins have been used as topical pesticides to protect crops, and more recently the proteins have been expressed in transgenic plants to confer inherent pest resistance. Bt transgenic crops have been overwhelmingly successful and beneficial, leading to higher yields and reducing the use of chemical pesticides and fossil fuels. However, their deployment has attracted some criticism particularly with regard to the potential evolution of pest-resistant insect strains. Here, we review recent progress in the development of Bt technology and the countermeasures that have been introduced to prevent the evolution of resistant insect populations.

The potential impact of plant biotechnology on the Millennium Development Goals.

The eight Millennium Development Goals (MDGs) are international development targets for the year 2015 that aim to achieve relative improvements in the standards of health, socioeconomic status and education in the world's poorest countries. Many of the challenges addressed by the MDGs reflect the direct or indirect consequences of subsistence agriculture in the developing world, and hence, plant biotechnology has an important role to play in helping to achieve MDG targets. In this opinion article, we discuss each of the MDGs in turn, provide examples to show how plant biotechnology may be able to accelerate progress towards the stated MDG objectives, and offer our opinion on the likelihood of such technology being implemented. In combination with other strategies, plant biotechnology can make a contribution towards sustainable development in the future although the extent to which progress can be made in today's political climate depends on how we deal with current barriers to adoption.

The regulation of carotenoid pigmentation in flowers.

Carotenoids fulfill many processes that are essential for normal growth and development in plants, but they are also responsible for the breathtaking variety of red-to-yellow colors we see in flowers and fruits. Although such visual diversity helps to attract pollinators and encourages herbivores to distribute seeds, humans also benefit from the aesthetic properties of flowers and an entire floriculture industry has developed on the basis that new and attractive varieties can be produced. Over the last decade, much has been learned about the impact of carotenoid metabolism on flower color development and the molecular basis of flower color. A number of different regulatory mechanisms have been described ranging from the transcriptional regulation of genes involved in carotenoid synthesis to the control of carotenoid storage in sink organs. This means we can now explain many of the natural colorful varieties we see around us and also engineer plants to produce flowers with novel and exciting varieties that are not provided by nature.

Cloning and functional characterization of the maize carotenoid isomerase and ß-carotene hydroxylase genes and their regulation during endosperm maturation.

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and ß-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9'-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert ß-carotene into ß-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form ß-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.

When more is better: multigene engineering in plants.

The genomics revolution has taught us that a great deal of information can be derived from studying many genes or proteins at the same time. We are beginning to see this approach blossoming in applied research. Instead of attempting to generate useful transgenic plants by introducing single genes, we now see an increasing number of researchers embracing multigene transfer (MGT) as an approach to generate plants with more ambitious phenotypes. MGT allows researchers to achieve goals that were once impossible - the import of entire metabolic pathways, the expression of entire protein complexes, the development of transgenic crops simultaneously engineered to produce a spectrum of added-value compounds. The potential appears limitless.