CLEANING-UP CROP GENOMES THROUGH INTRAGENIC MODIFICATION
Caius Rommens
September, 2007

Awareness that fruits and vegetables are excellent sources of nutrients gradually evolved during the last three centuries and is currently reinforced through USDA-backed promotion programs such as "5 A Day." However, there are still many issues associated with today’s food crops. Two recent reviews discuss the importance of removing lingering toxins and allergens while enhancing the levels of health-promoting antioxidants1,2. Such improvements may be accomplished efficiently through intragenic modification, a new approach to genetic engineering that transforms plants with native genetic elements only.

Today’s crops as a work-in-progress
Crop improvement programs are primarily directed toward traits that have always been at the forefront of the breeder’s mind: ever more yield with, in some cases, a little added taste. In an often desperate effort to accomplish these goals effectively, breeders create as much DNA diversity as possible. They recombine entire genomes of cultivated crops with those of wild relatives to capture at least some of the diversity that evolved within species boundaries. Additionally, genes are modified or inactivated through chemical mutagenesis programs that induce extensive substitutions, deletions, or rearrangements. New varieties derived from introgression or mutation breeding may, indeed, display powerful new trait combinations. However, they also express many less-desirable characteristics that were not considered during the selection process (Fig. 1).

Genomes of crops such as peanut, wheat, soybean, rice, and apple are peppered with allergen-encoding genes. Transfer of these genes from existing to new varieties is often considered inevitable. Consequently, consumption of a single peanut can be life-threatening to people predisposed to developing allergic reactions, and bread intake continues to damage the intestinal lining of about 0.8% of Americans who suffer from Celiac disease. Food crops also contain numerous genes involved in the biosynthesis of natural toxins and antinutritional compounds, including glycoalkaloids, cyanogenic glycosides, glucosinolates, coumarins, and gossypol. Indeed, the majority of pesticides in the human diet are naturally produced in the edible parts of crops, often at levels at or near those known to cause health issues3. Additional toxins may be produced upon heat processing. High levels of asparagine and reducing sugars in potato tubers and wheat flour trigger the heat-induced formation of the toxic compound acrylamide4. Furthermore, the polyunsaturated fatty acids in frying oils are rapidly oxidized upon heating to produce carcinogens.

The functional activity of toxin- and allergen-associated genes is contrasted by the frequent inactivity of potentially beneficial genes. For instance, crops such as tomato and potato express the key gene in flavonol biosynthesis in anthers but not in fruits and tubers, respectively. This tissue specificity limits the dietary availability of some of the most powerful antioxidants. Similarly, levels of carotenoids, anthocyanins, and phenolic compounds are often much lower than could potentially be attained.

Intragenic modification as a new extension to plant breeding
The genetic complexity of most undesirable features complicates efforts to eliminate them systematically through traditional breeding. Furthermore, it is difficult to enhance food quality without compromising yield. Many genes associated with the biosynthesis of toxins play an important role in the plant’s physiology and cannot be simply knocked-out. The easiest route to carefully modifying the expression levels of specific genes is afforded by genetic engineering. Unfortunately, the last twelve years have only yielded a one-sided approach to this technology. A few large agricultural biotechnology companies established a near-monopoly position on commercial applications, which were directed toward the permanent incorporation of bacterial, viral, and synthetic DNA into crops. Although the resulting varieties displayed high levels of herbicide tolerance and insect resistance, their release into the environment triggered widespread biosafety and ethics concerns. In the United States, public support for genetic engineering is still at the same low levels (26 – 27%) as in 2001 (http://pewagbiotech.org). This lukewarm response provided the backdrop for non-governmental organizations (NGOs) such as Greenpeace to successfully discourage the production and sale of genetically engineered specialty crops.

In 2003, Kaare M. Nielsen (University of Tromsø, Norway) proposed to bridge the gap between agricultural biotechnology companies on one side and consumers and NGOs on the other by diversifying genetically engineered crops based on the genetic distance between DNA source and target crop5. He defined organisms transformed with foreign DNA as transgenic, while using the term intragenic for plants containing native DNA. Intragenic modification isolates specific genetic elements from a plant, recombines them in vitro, and inserts the resulting expression cassettes into a plant that belongs to the same sexual compatibility group using plant-derived transfer (P-) DNAs and marker-free transformation6. This new approach to genetic engineering improves the agronomic performance or nutritional characteristics of crops but does not introduce traits that are new to the sexual compatibility group. Intragenic modification could also be applied to eliminate numerous allergens or toxins by silencing the associated genes. For instance, the down-regulated expression of potato genes involved in starch degradation resulted in a dramatic reduction of acrylamide levels in french fries7. Other examples of intragenic modification relate to the many traits that can be enhanced through all-native DNA transformation (Fig. 2).

In contrast to traditional plant breeding, intragenic plants lack new unknown DNA that may comprise genes associated with the production of toxins, allergens, or anti-nutritional compounds. The modified plants also lack selectable marker genes, powerful insecticidal genes, or any other foreign genes that are new to agriculture or the food stream. Furthermore, the modified expression levels of one or several native genes are expected to trigger phenotypic, biochemical, or physiological variations that already evolved within the sexual compatibility group. One argument for this assertion is that any modification accomplished through all-native DNA transformation could, at least theoretically, be created by recombination. At one end of the spectrum are the knock-out or loss-of-function mutations that can be isolated for many non-essential genes in natural populations, and are obtained at higher frequency using either natural or chemical mutagens. Individuals with enhanced gene expression, at the other end of the spectrum, may be recovered during plant selection, such as those adapted to specific environmental stresses. Both classes yield rare phenotypes pursued by breeders that can often be developed using intragenics. In a targeted analysis of important compounds and metabolites in genetically modified potato tubers with altered primary carbohydrate metabolism, polyamine biosynthesis, and glycoprotein processing demonstrated that there were no consistent differences with respect to appropriate controls8. Broader scale metabolomic and proteomic analyses reached a similar conclusion9. Thus, intragenic modification provides an effective means of enhancing the value of food crops in sustaining and enhancing health, while avoiding issues associated with the traditional breeding and transgenic approaches.

References

1. Rommens CM (2007) Intragenic crop improvement: combining the benefits of traditional breeding and genetic engineering. J Agric Food Chem 55, 4281-4288

2. Rommens CM et al. (2007) The intragenic approach as a new extension to traditional plant breeding. Trends Plant Sci, in press

3. Ames BN, Profet M, Gold LS (1990) Nature’s chemicals and synthetic chemicals: comparative toxicology. Proc Natl Acad Sci USA 87, 7782-7786

4. Tareke E et al. (2002) Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J Agric Food Chem 50, 4998-5006

5. Nielsen KM (2003) Transgenic organisms: time for conceptual diversification? Nat Biotechnol 21, 227-228

6. Rommens CM et al. (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol 135, 421-431

7. Rommens CM et al. (2007) Improving potato storage and processing characteristics through all-native DNA transformation. J Agric Food Chem 54, 9882-9887

8. Shepherd LV et al. (2006) Assessing the potential for unintended effects in genetically modified potatoes perturbed in metabolic and developmental processes. Targeted analysis of key nutrients and anti-nutrients. Transgenic Res 15, 409-425

9. Lehesranta SJ et al. (2005) Comparison of tuber proteomes of potato varieties, landraces, and genetically modified lines. Plant Physiol 138, 1690-1699

10. Muir SR et al. (2001) Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nat Biotechnol 19, 470-474

11. Ralph J et al. (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol Chem 281, 8843-8853

12. Liu Q, Singh SP, Green AG (2002) High-stearic and High-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol 129, 1732-1743

Caius Rommens
Simplot Plant Sciences
J. R. Simplot Company
Boise, ID
crommens@simplot.com