One of the principal aims of genetic modification in plants is to confer
resistance against pests and diseases in order to increase crop yields.
However, even in successful transgenic plants, the issue of biosafety must
be addressed, to ensure that no unintended biological modifications are
introduced, and to calm public fears. Even though only one or two genes
might be modified in a plant, it cannot be assumed that they will not modify
the protein profile. Any such alterations could lead to undesired plant
metabolites, including potentially toxic compounds.

Recent comparative studies on the proteome and transcriptome of several
transgenic plants, including the tomato, potato and soybean, have found very
few differences between the modified and wild-type forms. In fact, fewer
differences were found than between plant varieties. However, a group of
Italian researchers recently reported that the introduction of a foreign
gene coding for the signalling peptide systemin into tobacco plants brought
about marked changes in the tobacco proteome. So, each transgenic
modification should be examined case by case.

Now, that same Italian group has examined the proteomes of transgenic
plants, generated by Agrobacterium tumefaciens-mediated transformation, that
were designed to offer antiviral resistance. Angiola Desiderio and
colleagues from Genetics and Plant Genomics Section of ENEA in Rome and the
Proteomics and Mass Spectrometry Lab of ISPAAM in Naples compared the
protein profiles of two plants expressing different recombinant antibodies
with those of their unmodified forms.

The tomato Lycopersicon esculentum cv. MicroTom was modified to express the
antibody scFv(G4) against the cucumber mosaic virus (CMV) coat protein. The
herbaceous plant Nicotiana benthamiana, which is closely related to tobacco,
was engineered to express the antibody scFv(B9) against the G1 envelope
protein of tomato spotted wilt virus. These viruses both contribute to large
crop losses.

Seven protein extraction procedures were tested for both plants, to ensure
the optimum number of proteins was isolated and detected. The presence of
compounds such as pigments, plant metabolites and polysaccharides have
detrimental effects on gel electrophoresis so every effort was made to
remove them. The selected protocol used trichloroacetic acid/acetone rather
than phenol to extract proteins from the plant leaves.

The differences in protein profiles between the transgenic and control
plants were highlighted by two-dimensional differential gel electrophoresis,
using imaging techniques and a statistical analysis to identify proteins
that were differentially expressed. A total of 1818 spots were detected on
the tomato gels but only 10 were differentially expressed. Similarly, 8
proteins out of 1989 for N. benthamiana were apparently affected by genetic
transformation. However, the differences in expression were low with an
average ratio of less than 2.4.

The proteins were identified by mass spectrometric techniques. The majority
were single expression products involved in photosynthesis or defence
processes rather than metabolic pathways, so could be caused by "minimal
environmental stimuli." Taken together with the low expression ratios, the
data led the researchers to conclude that "the proteomic differences
observed between transgenic and control plants are negligible, defined and
more likely due to physiological variations." This conclusion is consistent
with published data for genome transformation performed by Agrobacterium
infection.

The expression levels of the scFv antibodies were so low that they were
undetectable by 2D gel electrophoresis, although weak signals were observed
by Western blotting. These low levels, less than 0.005% of the total soluble
protein, were nevertheless still sufficient to confer virus resistance for
at least 60 days after inoculation. Untransformed tomato plants were
severely affected within 18 days.

The results confirm the close similarity of the protein profiles of both
plants and show that virus protection can be conferred safely by genetic
engineering. The same confirmatory procedure should be carried out on every
new transgenic plant, since their protein profiles and metabolisms are all
unique, to ensure they can be used safely and effectively.

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