www.checkbiotech.org ; www.raupp.info ; www.czu.cz

It is generally accepted that traditional food is safe for the majority of
consumers. For the introduction of a new variant or cultivar developed from
a traditional crop plant, maximum limits have been set in some cases, e.g.,
for potato and oilseed rape, to the content of known toxins, January 2006 by
Sirpa O. K?¤renlampi and Satu J. Lehesranta.

The requirements are much more stringent if the crop is developed by using
genetic engineering. Why is it so? In a majority of cases seen so far, a new
gene, often derived from other plants or microbial species, has been
introduced to a non-predetermined location in the plant genome. It is quite
feasible to ask the question whether the new gene products are safe or not.
Therefore, for all genetically modified crop plants, the safety of the newly
introduced proteins needs to be demonstrated before the plants can be
released into the market.

Another point of concern is the random integration of the new gene into the
plant genome. Both the new gene itself and its site of integration may give
rise to unintended adverse effects. For example, transgene integration might
interrupt regulatory sequences or open reading frames leading to novel
fusion proteins and, thereby, modify plant metabolism. These modifications
could compromise the safety of the food crops by, for instance, leading to
the production of new allergens or toxins. Having the gene and the
integration site well characterised should provide a good basis for the
safety assessment.

However, it is a common practice today to perform a large number of
analyses, so-called targeted analyses, to demonstrate that the
characteristics of the novel crop are comparable with those of the
conventional counterpart, in addition to the intended alterations. Targeted
analyses include key macronutrients, micronutrients, antinutrients, and
toxins. In certain cases, toxicity studies on experimental animals are
advised. And yet, the question about the unintended effects does not seem to
be covered in a way that would escape all criticism.

Cellini et al. have considered transgene integration in the context of
naturally occurring DNA recombination. It is well known that genetic
variation is the cornerstone of plant breeding. Natural chromosomal
recombination plays a central role in generating new variation.
Non-homologous end joining, which is the predominant form of recombination
in plants, rarely occurs without any sequence alterations, and usually gives
rise to deletions of up to more than 1 kb and introduction of new filler
DNA. Since the double-strand break repair system involved in recombination
is more error-prone in plants than in other organisms, errors that change
the original sequence occur at a very high frequency. The fact that
gene-rich regions (and genes) are hotspots for recombination has facilitated
the emergence of novel characteristics in crop plants.

Integration of exogenous DNA (transgene) occurs via the same mechanism as
natural recombination. Several types of rearrangements are thus observed,
both in transgene integration sites and in natural recombination sites.
While this mechanism provides a selection of natural variation for breeders,
it is also a source of unintended effects similar to that in genetically
engineered crop plants.

In the light of variation generated by natural recombination and by the
repertoire of conventional breeding technologies exploited for decades, the
question is how much variation in the overall genetic makeup of a crop plant
might be generated by the transfer and integration of a single gene,
compared to the variation already existing. A related question is how
probable are the unintended effects that extend beyond this variation.

To answer these and other questions, we made a comparative analysis of eight
GM lines of potato, including vector-only lines without the target gene. The
parent cultivar, Desir?Še, and a line that had undergone tissue culture
only, were included as non-GM comparators. Nine of 730 proteins showed
statistically significant differences among the GM lines and controls. No
new proteins that would be unique to the individual GM lines were observed.
The conclusion from this study, supported by the EU-funded GMOCARE project,
was that there was no evidence for any major changes in protein patterns of
the GM lines tested.

It can be argued that proteomics is not sensitive enough to find differences
between potato lines or varieties. The European breeders have developed a
large number of very different potato cultivars, many of them with genes
introgressed from other Solanum species. Of that diversity, we analysed 32
non-GM potato genotypes, including 21 conventional cultivars, eight
landraces, and three lines of S. phureja. From that study it was obvious
that there is a great deal of variation in the protein patterns of the
different potato genotypes: out of 1111 protein spots analyzed, 1077
differed significantly among two or more genotypes. The protein profile of
the diploid species S. phureja could be clearly distinguished from the ones
of the tetraploid S. tuberosum genotypes.

These studies indicated that the variation between the non-GM
cultivars/genotypes was much greater than the differences between the GM
lines. This was further confirmed by direct comparison of some of the GM
lines with two non-GM genotypes; there was no separation among the GM lines
and their control, but the two non-GM genotypes separated very clearly from
each other and from all Desir?Še-based lines. In other words, there were
considerably fewer differences between the GM and non-GM lines of the same
genetic background than between different non-GM cultivars. Many of the
proteins that contributed to the separation of the non-GM genotypes appeared
to be involved in disease and defense responses, sugar and energy
metabolism, or protein targeting and storage, and are presently considered
to convey no safety risk.

Our results have been corroborated recently by Catchpole et al., who
compared several GM potato lines and cultivars using metabolic profiling.
The authors found differences between the GM lines only in those metabolites
that were targets of the genetic modification; apart from those compounds,
the GM lines could not be distinguished from their controls. On the other
hand, all cultivars could be clearly distinguished from one another.

The results of both profiling studies are not surprising, considering what
is now known about the nature of plant genome and its dynamics. Even though
genetic modification does not generate major changes apart from the ones
targeted, a protein identified at an increased level in the GM line compared
to the conventional counterpart might be worth further attention if the
level clearly falls outside the normal variation. This is to exclude any
risks from, for example, potent allergens. As current profiling methods
produce a huge amount of data, it is almost inevitable that some
statistically significant differences will be found. Therefore the focus
should be in truly consistent differences.

How feasible are profiling techniques in general as tools to provide
additional data for the risk assessment of GM crops? Do they provide added
value worth the investment? Do they give reassurance that unintended adverse
effects have not occurred? Non-targeted methods, such as transcriptional,
protein, and metabolite profiling, offer potentially unbiased approaches to
the detection of unintended effects. Of these, transcriptomics is possibly
the most comprehensive, with full genome arrays currently available for a
limited number of plant species.

While it is clear that a comprehensive coverage of all proteins and
metabolites present in a given tissue is difficult to obtain with current
technologies, proteins are the key molecules of interest, as they are
potential allergens and catalyse the synthesis of metabolites, some of which
are potential toxins.

To assess observed differences within the context of natural variation in
composition, comparative data of 'normal' protein levels are needed to
understand the effect of genetic background, developmental stages,
physiological states, environmental conditions, and cultivation techniques,
and to be able to set the criteria against which a determination of a
significant difference worth considering as a possible safety risk can be
made. Currently there is very little information publicly available on
protein patterns in potato tubers or in any other crops.

As with other profiling methods, proteomic screening is not yet routine for
assessing the safety of GM products. However, proteomic profiling has the
potential to reduce uncertainty by providing much more information about
crop composition than does targeted analysis alone, especially in
combination with other profiling methods. In addition, multivariate
statistical methods can give a much better overall picture of how the given
samples relate to each other than does the comparison of single compounds.
These facts may make proteomics increasingly important when developing
second generation GM crops with multiple genes, engineered metabolic
pathways, or edible pharmaceuticals.

References:

Kuiper HA, Kleter GA, Noteborn HPJM & Kok EJ (2001) Assessment of the food
safety issues related to genetically modified foods. Plant J 27, 503-528

Cellini F et al. (2004) Unintended effects and their detection in
genetically modified crops. Food Chem Toxicol 42, 1089-1125

Lehesranta SJ, Davies HV, Shepherd LVT, Nunan N, McNicol JW, Auriola S,
Koistinen KM, Suomalainen S, Kokko HI, & K?¤renlampi SO (2005) Comparison of
tuber proteomes of potato (Solanum sp.) varieties, landraces and genetically
modified lines. Plant Physiol 138, 1690-1699

Catchpole GS, Beckmann M, Enot DP, Mondhe M, Zywicki B, Taylor J, Hardy N,
Smith A, King RD, Kell DB, Fiehn O & Draper J (2005) Hierarchical
metabolomics demonstrates substantial compositional similarity between
genetically modified and conventional potato crops. Proc Natl Acad Sci USA
102, 14458-14462

Sirpa O. K?¤renlampi and Satu J. Lehesranta
Institute of Applied Biotechnology, University of Kuopio
FIN-70211 Kuopio, Finland
skarenla@messi.uku.fi

[www.isb.vt.edu]

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