Wednesday, April 6, 2011

By Briardo Llorente, Fernando Bravo-Almonacid, Héctor N. Torres, Mirtha M. Flawiá and Guillermo D. Alonso

The creation of the first genetically engineered (GE) organisms in the 1970s initiated a still ongoing debate regarding their safety. Despite this concern, GE crops have been increasingly adopted worldwide, and the global area of GE crops has risen from 1.7 million hectares in 1996 to 134 million hectares in 20091.
Regulatory authorities determine the safety of GE crops using a “comparative safety assessment” strategy. This approach is intended to define the substantial equivalence of GE crops; that is, to compare the phenotype and composition of GE crops to parental or closely related counterparts with an established history of safe use for differences and undesired side effects derived from the genetic modification2.

This evaluation model has a clean safety record, proving satisfactory for assessing the safety of so-called first-generation GE crops with resistance to pests or tolerance to select herbicides, and without major metabolic and compositional alterations. However, the advent of increasingly complex biotech crops has led to widespread controversies regarding the future suitability of current safety assessment procedures.

Consumers increasingly demand improved food quality; hence researchers are developing next-generation GE crops to provide food with enhanced quality or tailored properties to match these demands. Consequently, in addition to securing permits, the commercialization of crops with value-added traits such as improved nutrition or food functionality should involve earning public acceptance. Scientific studies demonstrating the benefits of GE quality-enhanced crops—not just speculations—could positively impact public perception. Additionally, because sensory qualities can be an important determinant of the public acceptance of new foods, assessing consumer perception of the GE crops should be included in the development of quality-improved crops.

The scientific community, regulatory authorities, and the general public are paying close attention to these issues,
as next-generation biotech crops are being developed. This review provides a brief overview of some recent studies in which these topics were addressed by applying different approaches in GE potato, tomato, carrot, and lettuce cultivars.

Holistic assessment of complex biotech crops

Chakraborty and collaborators3 from India recently completed a broadly integrated analysis of potato plants engineered to express in tuber the seed storage protein AmA1 (Amaranth Albumin 1) from amaranth, a South American plant widely eaten across the humid tropics. Even though no visible phenotypic changes in the morphology of the engineered plants were observed, chemical (Kjeldahl method), HPLC (High-Performance Liquid Chromatography), and 2D Gel Electrophoresis analyses revealed that the proteome was rebalanced in the GE tubers, resulting in up to a 60% increase in total protein and a significant increase in several essential amino acids in comparison to wild-type (WT) controls. Furthermore, some unexpected alterations, such as enhanced photosynthetic activity and an increase in tuber yield, were detected in the GE plants under field conditions.

Chakraborty and co-workers performed in silico and in vitro analyses and in vivo studies with experimental animal models, which indicated these GE tubers are nonallergenic and nontoxic. In addition, the authors determined that the cooking and processing qualities of the GE tubers were better than or as good as WT tubers. Together, this work represents a very good example of translational research intended to improve crop nutritional value in which the safety and quality properties of the GE crop have also been explored.

In another recently published study, Llorente and colleagues4 from Argentina performed, as a proof of concept, a comparative safety assessment on a quality-improved biotech crop—potato plants engineered for reduced tuber browning. The researchers silenced, in the GE potato, a family of genes coding for polyphenol oxidase (PPO), an enzyme with yet unknown biological function but known to be responsible for the enzymatic browning reactions typically observed in damaged plant tissue.

Under glasshouse conditions, Llorente and colleagues characterized the GE potatoes at both physiological and  molecular levels and concluded that the potatoes were equivalent to WT plants when yield-associated traits and photosynthesis were evaluated. On the other hand, primary metabolism analysis performed with a combination of spectrophotometric and GC-MS (Gas Chromatography coupled to Mass Spectrometry) techniques revealed that the transgenic tubers had several unanticipated metabolic alterations when compared to the WT controls.

GE lines were also tested in silico and through in vivo analyses in mice to look for unintended allergenic, toxicological, and nutritional effects; no unfavorable results were found. These results suggest that the next generation of biotech crops can be properly assessed following the existing evaluation criterion.

Interestingly, during the feeding trials, mice fed a diet supplemented with nonbrowning potatoes consumed more potatoes than mice fed a diet supplemented with WT potatoes. Even though the basis of these results was not established in this paper, the authors speculated that differential organoleptic and palatability properties between the GE and WT tubers, originating from dissimilarities in metabolite content together with the slower oxidative deterioration of the GE tubers, could account for the observed results.

Consequently, in a subsequent study5, the same group investigated the role potato aroma may have in mice preference for the nonbrowning potatoes. Applying a set of widely used behavioral paradigms from the neurobiological sciences field, the authors performed experiments with both mice and humans.

Aroma preferences in mice were estimated by how much time they spent exploring two food containers in an open field paradigm or a unique food container in a hole-board paradigm. When the potato samples were fresh, with insufficient time to turn brown, the mice showed no preference between WT and GE tubers. But quite surprisingly, when the potato samples aged for 24 hours, allowing time for oxidation, mice consistently showed a stronger preference for the aroma of the GE potatoes.

Results from human olfactory analyses of WT versus GE tubers were similar to those obtained from mice. Participants subjected to a triangle test were able to discriminate the WT from the GE potatoes considerably better when the potato samples were aged. In addition, even though the WT and GE potatoes smelled equally pleasant to human participants, they reported that the nonbrowning potatoes had a more intense and familiar aroma. This report represents the first successful example of the implementation of an animal model and neurobiological paradigms in the study of a genetically engineered crop. Even though Llorente and colleagues conducted these studies under laboratory conditions, these reports embody a comprehensive examination in which both safety and sensory issues were assessed in a complex GE crop with improved quality and metabolic alterations.

At least three other recent studies have performed sensory analyses of complex GE crops. Davidovich-Rikanati et al.6, in a cooperative work between Israeli and American teams, modified the flavor and aroma of tomato fruits by expressing the geraniol synthase gene (GES) from Ocimum basilicum (sweet basil) under the control of a ripening-specific promoter. The expression of GES caused marked changes in the volatile compounds profile of these GE tomatoes; particularly monoterpene contents were enhanced at the expense of precursors for lycopene and phytoene biosynthesis, which were reduced. When smell and taste sensory trials were performed, most of the participants judged that the GE tomatoes were more aromatic and also preferred them over the WT controls.

In a very interesting study performed in USA, Rommens and co-workers7 reported they genetically engineered high-antioxidant potatoes by simultaneously overexpressing a modified MYB transcription factor gene (StMtf1M) and silencing the flavonoid-3′,5′-hydroxylase gene (F3′5′h). The GE tubers contained massive amounts of the health-promoting compound kaempferol (almost 100-fold increase) and also accumulated fourfold increased levels of antioxidant phenolic acids. Even though the GE lines produced normal tuber yields, microarray analyses of gene expression demonstrated that several groups of genes were altered in comparison to the WT lines. The authors found that the high levels of phenolic antioxidants persisted in the GE tubers after cooking and, by performing sensory analyses with a panel of food scientists, also demonstrated that the texture and taste of the GE potatoes were indistinguishable from that of the untransformed controls.

Using a similar approach, Park et al.8, from the USA, convened a panel of highly trained descriptive experts to evaluate the quality properties of GE biofortified lettuce plants engineered to express the Arabidopsis thaliana sCAX1 calcium transporter gene. The biofortified lettuce lines presented normal morphology, growth, total yield, and seed set, but contained up to 32% more calcium than WT control plants. However, in spite of this compositional modification, the panelists found no significant differences in the flavor, bitterness, or crispness of the GE lettuce.

Previously9, using a dual-stable isotope method and feeding studies, the same group demonstrated that both mice and humans had significantly increased total calcium absorption when fed GE sCAX1-expressing carrots,
validating the functionality of this biotechnological strategy.

The potential impact of GE crops on promoting health benefits was recently evaluated in a revealing study performed in Europe. Butelli and colleagues10 expressed in tomato two transcription factors from snapdragon (DEL and ROS1) with the aim of improving its antioxidant properties. The resulting GE tomatoes developed a purple coloration and accumulated increased amounts of antioxidant compounds such as anthocyanins and flavonols. Remarkably, cancer-susceptible mice (p53-knockout mice) fed diets supplemented with the GE tomatoes showed a significant extension of life span when compared with mice fed standard diets or diets supplemented with WT tomatoes.

This study denotes the potential health-promoting or disease-preventing effects that can be achieved with next-generation GE crops.

Collectively, these motivating studies are examples of how the development of genetically engineered improved crops can be assessed in a holistic manner to obtain a broader understanding of their commercial viability.

Perspectives

A promising variety of genetically engineered crops displaying enhanced quality traits with benefits to both producers
and consumers is being developed worldwide. Their acceptability will require them to be safe and in harmony with consumers expectations.

In agreement with other scientists11, our perception is that the regulation of new crop varieties should be performed in a case-by-case manner, based not according to how they are bred, but to their novelty and to humanitarian needs.

Looking to the future, scientists working in the field of plant biotechnology might have to deal with the dual challenge of meeting consumer demands and building public trust about genetic engineering technology. Establishing
safety, sensory, and functionality evaluations as part of the integrative development of genetically engineered crops might be a fruitful way to meet these challenges. Certainly, scientific studies dealing with these subjects are the order of the day and will contribute to public awareness about the safety and benefits of GE crops grounded on science.

References
1. James C. Global Status of Commercialized Biotech/GM Crops: 2009. ISAAA Brief No. 41. ISAAA: Ithaca, NY. (2009)
2. Kok EJ & Kuiper HA. Comparative safety assessment for biotech crops. Trends Biotechnol 21(10), 439-444 (2003)
3. Chakraborty S, et al. Next-generation protein-rich potato expressing the seed protein gene AmA1 is a result of proteome rebalancing in transgenic tuber. Proc Natl Acad Sci USA 107(41), 17533-17538 (2010)
4. Llorente B, et al. Safety assessment of nonbrowning potatoes: opening the discussion about the relevance of substantial equivalence on next generation
biotech crops. Plant Biotechnol J 9(2), 136-150 (2011)
5. Llorente B, et al. Improvement of aroma in transgenic potato as a consequence of impairing tuber browning. PLoS One 5(11), e14030 (2010)
6. Davidovich-Rikanati R, et al. Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nat Biotechnol 25(8), 899-901(2007)
7. Rommens CM, et al. Engineered native pathways for high kaempferol and caffeoylquinate production in potato. Plant Biotechnol J 6(9), 870-886 (2008)
8. Park S, et al. Sensory analysis of calcium-biofortified lettuce. Plant Biotechnol J 7(1), 106-117 (2009)
9. Morris, et al. Nutritional impact of elevated calcium transport activity in carrots. Proc Natl Acad Sci USA 105(5), 1431-1435 (2008)
10. Butelli E, et al. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26(11), 1301-1308 (2008)
11. Potrykus I. Regulation must be revolutionized. Nature 466(7306), 561 (2010)

Briardo Llorente1*, Fernando Bravo-Almonacid1,2, Héctor N. Torres1, Mirtha M. Flawiá1, and
Guillermo D. Alonso1
1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, CONICET and FCEyN, Universidad de Buenos Aires Buenos Aires, Argentina
2 Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina
* llorente@dna.uba.ar