Modulation of regulatory control proteins and regulatory processes has occurred during plant domestication through both natural and selected breeding of improved crop varieties.128131 For example, the changes responsible for improved wheat yields as part of the "green revolution" involved selection for mutant Reduced height-1 genes through conventional breeding.132 The proteins encoded by these genes are regulators of endogenous gene transcription that make wheat plants insensitive to giberellin, a plant growth regulator, thus making the plants shorter and protecting them from collapsing under their own weight.132 As a consequence, yield is increased at harvest. Wheat domestication also involved the Q gene, an AP-2-like transcription factor that confers free-threshing character and reduces fragility, enabling more efficient grain harvesting.133 The domestication of maize from its ancestral form, teo-sinte, has involved selection for enhanced expression of the teosinte branched 1 transcription factor134 and regulatory changes in the maize allele of the teosinte glume architechture transcription factor.135 Another example of the impact of transcription factors in corn breeding is a mutation in the opaque 2 transcription factor. This mutation led to the generation of Quality Protein Maize (QPM), an improved nutrition maize variety (high in lysine content) that was the winner of the World Food Prize in 2000.136 Reduced grain shattering resulting from a single base pair mutation in the DNA binding domain of the putative transcription factor sh4 has been thought to be a key event in the domestication of rice.137 Tomato hybrid cultivars with a mutant transcription factor yield fruit with a longer shelf life.138
We are now learning that the domestication and breeding of modern crops with beneficial traits carried out over the past centuries has involved selection for changes in proteins regulating endogenous plant gene expression. Transcription factors have played a prominent role in these processes. These crop varieties produced as a result of altered transcription factor expression have an established history of safe consumption as they are staples in the human diet. This demonstrates that plants with alterations in endogenous gene expression of proteins that modulate other endogenous plant genes have been safely consumed.
Profiling technologies such as genomics, proteomics, and metabolomics have facilitated identification of genes that regulate endogenous plant processes and the phenotypic effects elicited by their protein products.139 Therefore, proteins that affect endogenous pathways are among the likely targets to improve the next generation of biotechnology-derived crops. During the last few years, there has been a growing number of biotechnology-derived plants with modifications in endogenous transcriptional regulatory processes.140-142
A fundamental principle to consider when evaluating the safety of these biotechnology-derived crops is that the transcription factor proteins operate through regulation of endogenous plant processes. Thus they are unlikely to produce novel metabolites not previously present in plants. These proteins will be structurally or functionally homologous to endogenous plant transcription factor proteins. They could also be obtained from the same crop into which they will be reintroduced through biotechnology.
During the growing season, plants are normally subjected to a variety of biotic and abiotic stress conditions. In response to these environmental conditions, a variety of transcription factor-mediated changes in endogenous plant gene expression occur. Humans and animals consume food or feed from crops that contain the cumulative gene expression changes that occur in plants grown under variable stress conditions.
There is a history of consumption of transcription factors as they are present in all eukaryotic cells, some of which are consumed as food. Out of an estimated 59,000 genes in the rice genome, approximately 1600 (~3%) are predicted to encode transcription factors.143 The soybean genome is predicted to contain approximately 1300 transcription factors out of an estimated 63,500 genes, representing about 2% of the genome.144 Questions concerning the safety of food or feed derived from crops containing introduced transcription factors should be considered in the context of the history of safe consumption of food and feed derived from plants containing these naturally and regularly occurring changes in transcriptional profiles.
An additional exposure consideration for many regulatory proteins is that they usually have a small number of specific targets. Moreover, although transcription factors are expressed in every cell, they are generally present in low levels in plant and animal tissues. In Arabidopsis, for example, the number of mRNAs encoding an individual transcription factor has been reported to range from 0.001 to 100 copies per cell, illustrating the relatively low level of these transcripts in plant cells.145 The wide range in potential levels for a given transcription factor may result from spatial (cell type), temporal (cell cycle), and developmental (life cycle) regulation of gene expression.141 Transcription factor proteins also tend to be present at very low amounts in plant tissue. For example, only 50 ^g (80 pmol) of KAP-2 transcription factor was obtained from 6 kg of bean cells, corresponding to about 8 ng of transcription factor protein per gram of tissue.146
Even with large uncertainties in available estimates, it is apparent that transcription factors represent only a tiny fraction of total plant proteins, and their concentrations (~ppb) are likely to be several orders of magnitude lower than proteins introduced into biotechnology-derived crops (ppm) to date (Table 11.3) or typical food proteins that might constitute 1% (10,000 ppm) or more of the total protein present in the food.16 Total protein levels in food crops can range from 10% for maize to 40% for soybeans.147 Tissues consumed from food animals also provide a dietary source of transcription factors and other regulatory control proteins as they are ubiquitous in the cells of animals, albeit at low levels. If levels of these transcription factors or other regulatory control proteins are elevated in food or feed beyond that normally observed in the plant product, this information would also be used in the evaluation of the history of safe consumption of related proteins.
The assessment of potential oral activity for introduced transcription factors needs to take into consideration the following factors:
1. The lack of a specific transport system for regulatory control proteins may provide an explanation, in part, as to how GI tract epithelia are continuously exposed to these proteins from dietary sources (plant- and animal-derived foods) without any evidence of biological response in mammals.
2. Transcription factors and many other proteins that regulate gene expression function in the nucleus. In order for ingested regulatory control proteins to be active in the consuming organism, the protein would thus need to not only survive digestive barriers, gain access to the systemic circulation, and be transported to a target tissue, but would also have to undergo cellular uptake, evade cytoplasmic degradation, and would require subsequent transport across the nuclear membrane and into the nucleus. Selective import of proteins across the nuclear membrane requires the presence of a nuclear localization signal within the protein sequence.148 Whether an exogenous transcription factor or other regulatory control protein would enter the nucleus would depend partly on the interaction between that protein and nuclear import machinery in cells of the consuming organism. The specificity required for such interactions adds yet another barrier to function of dietary proteins that regulate gene expression.
Based on all of the aforementioned considerations, one can conclude that the existing risk assessment procedures used to assess safety of proteins introduced into biotechnology-derived crops are also applicable to transcription factors.
Since endogenous metabolic pathways may be modified to achieve the desired plant improvement, the agronomic performance and phenotypic appearance of the plant will be examined under a variety of environmental conditions to confirm that there are no deleterious unintended changes. The composition of grain or seed will also be analyzed to confirm that endogenous nutrients or antinutrients have not changed, unless the intended technical effect results in changes in levels of endogenous nutrients. In this case, the safety and nutritional impact of those changes will be evaluated independently.
If there is evidence of significant unexpected/unintended molecular, compositional, agronomic, and/or phenotypic changes that could be adverse, then the safety implications of these changes would require further study before a decision could be made whether the crop could be safety used. This safety assessment process which is aligned with international guidelines discussed previously is considered to be fully adequate to confirm the safety of food/feed derived from plants whose metabolic pathways are modified to achieve intended improvements in the crop.
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