Naturally transgenic plants as a model for studying delayed environmental risks of GMO cultivation: new facts and generalizations.

Full Text

Introduction

Transgenic plants have become a significant aspect of modern global agriculture, as demonstrated by the annual increase in the area dedicated to genetically modified (GM) crops, which now exceeds 200 million hectares [1] (fig.1). Transgenic plants (GMOs) can help solve global problems because they have a number of significant advantages over traditional varieties and hybrids, including: resistance to pests and diseases, resistance to herbicides, increased yield and stability, improved nutritional and consumer qualities, resistance to abiotic stress [1-2]. This large-scale shift to biotechnological solutions has led the scientific community to focus not only on productivity but also on a thorough evaluation of potential environmental impacts. Key areas of concern include: 1) the effects of plant-produced insecticidal proteins on non-target organisms, such as beneficial insects and microorganisms in the rhizosphere and phyllosphere; and 2) the risk of uncontrolled transgene spread in the environment. This can happen through mechanical seed dispersal outside of agroecosystems and through cross-pollination between GM, traditional varieties, and their wild relatives, posing a threat of genetic contamination to natural populations [3]. The consequences of such flow can be evaluated by examining natural GMOs—plants whose ancestors underwent Agrobacterium-mediated transformation hundreds of thousands or even millions of years ago. Let's look at this topic in more detail.

Fig.1 Area under GM crops (a) and countries cultivating them (b)

Gene flow is a key ecological risk of GMO cultivation/

One of the key environmental risks associated with GM crops is transgene escape (also known as "gene flow" or "genetic pollution")—the uncontrolled spread of genetically engineered traits from intended crops into natural ecosystems or traditional farmland. The main causes of transgene escape can be categorized into two broad groups: biological mechanisms and agronomic/anthropogenic factors. Biological mechanisms include cross-pollination (via pollen) and the dispersal of vegetative parts and seeds [4-6]. The riskiest scenario involves cross-breeding with wild relatives [5-6]. If the region where the GM crop is grown contains wild or weedy relatives capable of cross-breeding, the transgene can "escape" into the wild population [7-8]. Agronomic and anthropogenic causes, including errors and accidents, involve mistakes occurring at various stages of agricultural production and regulatory violations. The consequences of transgene escape include the emergence of hybrids with increased resistance, which can become difficult-to-eradicate  weeds, a reduction in the genetic diversity of wild ancestors of cultivated plants, and changes in competitive relationships between species [3].

All of the above highlights the importance of conducting monitoring studies and developing strategies to prevent transgene leakage. However, current research is limited to only a few years of observation. In 2015, we proposed a model, based on naturally transgenic plants, for studying the delayed risks of GMO cultivation [9]. A substantial body of evidence has since accumulated, allowing us to revisit this topic.

Naturally transgenic plants

The term "naturally transgenic plants" refers to plants that have acquired foreign genes from a species other than their direct ancestors through a natural, horizontal gene transfer (HGT) process, without human intervention. The most famous and well-documented examples of naturally transgenic plants are species that were transformed by Agrobacterium/Rhizobium, the very same bacterium that scientists use as a tool to create transgenic plants in the lab [10]. Hundreds of species of naturally transgenic plants are currently known, and it is generally accepted that traces of Agrobacterium/Rhizobium -mediated transformation (called cT-DNA, cellular N-DNA) exist in the genomes of seven percent of dicotyledons [11-13]. The rapid development of next-generation sequencing and bioinformatics methods enables large-scale genome sequencing of various species within specific genera, providing invaluable data for assessing the diversity of natural transgenes. This analysis can be used to track the fate of natural transgenes in evolutionarily related plant groups [14-17].

Evolutionary fate of natural transgenes

Research conducted by our group shows that transgenes integrated into plant genomes millions or hundreds of thousands of years ago can follow varying evolutionary paths. Our analysis reveals that most natural transgenes accumulate mutations that render them nonfunctional, leading to their gradual deletion. In contrast, some natural transgenes persist under stabilizing selection, resulting in groups of species that retain functional transgenes [18-20].

Now, let's examine specific examples of natural transgenes with different evolutionary outcomes.

In a study by Bogomaz and co-authors [19], 23 species of the genus Arachis were characterized, with some species represented by various forms. Within this genus, virtually all studied accessions contained in their genomes at least one intact copy of a gene homologous to the cucumopine synthase gene. This gene catalyzes the production of cucumopine (fig.2), a low-molecular-weight compound utilized by some bacteria for nutrition. Additionally, fragments of genes were identified in some peanut genomes, whose Agrobacterium homologs encode enzymes involved in the sequential synthesis of mannopine and agropine. Notably, none of the species retained all three genes, and none of the detected fragments were intact. In 14 species, the genes encoding the mannopine-agropine synthetase enzymes were completely absent. It can be inferred that the cucumopine synthase gene was preserved under the influence of stabilizing selection, while the genes for other opines lost their significance for the ancestor of peanuts and were gradually eliminated.

Fig 2. Cucumopine and agropine synthesis reactions

Another interesting example involves representatives of the genus Vaccinium [20]. Among the 22 species studied within this genus and its related species, damaged copies of the transgene were predominant only in B. oxycoccos. In the other species, the transgenes remained intact. The mutations in the damaged gene consisted of extensive deletions. It is likely that mechanisms for eliminating foreign DNA have begun to operate within this species.

More extensive studies were conducted on plants from the Nicotiana and Camellia genera.

In the genus Nicotiana, eight distinct types of cellular T-DNA were analyzed [14-16]. Attempts to trace their phylogenetic relationships during speciation revealed that cultivated tobacco did not inherit all copies of T-DNA from a single ancestral genome. One T-DNA (TC) was completely lost.

In addition to TC, TE also experienced chromosomal rearrangements throughout its evolution [16].

Within the genus Camellia, 12 variants of cellular T-DNA have been described. The most ancient of these are TA and TD. Phylogenetic studies suggest that the ancestral form that gave rise to the sections Thea, Tuberculatae, Longipedicellatae, and Calpandria lost TD. The stages of occurrence of deletions within TA were reconstructed based on a comparative analysis of the TA insertion in C. fangchengensis and the majority of representatives of the Thea section. Modern TAs do not contain intact genes; however, the mutation pattern indicates that species from the Thea section and C. fangchengensis diverged from an intact sequence, subsequently accumulating point mutations, followed by an inversion and two deletions of TA fragments in C. fangchengensis.

Together, these data suggest that the plant eliminates non-functional sequences while preserving intact genes that confer a selective advantage.

Conclusion

Similar consequences cannot be ruled out for transgenic plants created by humans, especially given their uncontrolled spread in the environment. Brassica napus, Medicago sativa, and Agrostis stolonifera warrant particular attention in this context, as all three crops are found in the wild worldwide. Transgenic lines resistant to herbicides have been developed from each of these species. When exposed to a selective agent, transgenic forms and their hybrids gain an advantage over non-herbicide-tolerant plants [3].

Consequently, monitoring is essential to prevent adverse developments. In recent years, new information has emerged regarding the spread of transgenes through cross-pollination within a species and interspecific hybridization. Although interspecific hybridization occurs infrequently, it can have significant consequences for the fixation of transgenes within new genome [9]. The history of naturally occurring transgenic plants shows that unwanted transgenes can be eliminated from populations by removing the influence of the selective agent. This can be accomplished by abandoning the use of pesticides to which specific GMOs are resistant in favor of other pesticides to which the escaped GMOs are not resistant.

About the authors

Татьяна Валерьевна Матвеева

Author for correspondence.
Email: radishlet@gmail.com
ORCID iD: 0000-0001-8569-6665
SPIN-code: 3877-6598

department of genetics and biotechnology

References

Supplementary files