Why do plants need agrobacterial genes?

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INTRODUCTION

Agrobacteria are a group of soil bacteria consisting of representatives of several genera [1] able to transfer a fragment of their own DNA, called T-DNA (transferred DNA), and integrating it into the plant genome [2]. In the most cases, during such a transformation, only a part of the plant tissues is transgenic, as a rule, in the region of the root collar. Other tissues, including tissues of generative organs, remain nontransgenic [3].

There are examples of T-DNA occurring in the genome of a whole plant. When this happens, T-DNA is passed down for generations. Such T-DNA is called cellular T-DNA (cT-DNA), and plants containing it are called naturally transgenic or natural genetically modified organisms (nGMOs) [4]. In this study, we are dealing with horizontal gene transfer from agrobacteria to plants. The role of horizontal gene transfer in plant evolution has not yet been fully studied [5].

Based on our knowledge of the role of horizontal gene transfer in the evolution of prokaryotes, we can assume similar effects occur in eukaryotes, namely, the acquisition and inheritance of new traits that provide selective advantages, which can be divided into two groups:

1. improvement of existing functions, and
2. emergence of new functions in the recipient (for example, changes in nutrition, new protective functions) [6–8].

The first nGMOs resulting from ancient agrobacterial transformation were described in the genus Nicotiana L. [9], later they were detected by molecular genetic methods in two more genera, Linaria Mill. and Ipomoea L. [10–12]. In 2017, we published a review on the biological aspects of the naturally transgenic plants known at that time and the possible functions of genes obtained by plants from agrobacteria [13]. At that time, the functions of cT-DNA included the following:

• increase in root mass to adapt to plant growth in arid conditions,
• immunity to repeated agrobacterial infection,
• increased regenerative capacity,
• transition to earlier flowering and, as a result, transition to an annotinous life cycle, and
• influence on the communities of microorganisms of the rhizosphere and phyllosphere of the plant [13].

In recent years, the list of natural GMOs has increased substantially through the use of bioinformatic methods [14, 15]. Thus, the time has come for a new review of the functions of cT-DNA and the evolutionary role of the horizontal transfer of agrobacterial genes in plants. This review is focused on this subject.

New taxa of natural GMOs

The development of next generation sequencing methods has provided new opportunities for studying plant genomes, resulting in incremental growth of data on the their structure. Constantly updated databases are a valuable source for searching for new nGMOs [14].

Analysis of the sequenced genomes of land plants allowed us to identify homologues of agrobacterial genes only within the angiosperm division. At present, it is generally accepted that agrobacteria transform dicotyledons much more efficiently than they do monocotyledons [16]. This idea is supported by data on the distribution of natural GMOs. Among several dozen species of new natural GMOs, only two of them are monocotyledons. The systematic position of one of them (Dioscorea alata L.) is debatable, and the other species (Musa acuminata Colla) is assigned to natural GMOs only based on data obtained from the analysis of the root transcriptome [14]. Thus, these data require further research and careful interpretation.

Among the dicotyledonous plants, the confinement of naturally transgenic species to a specific taxonomic group was not noted. Natural GMOs are described within the orders Malpighiales, Fabales Rosales, Cucurbitales, Fagales, Brassicales, Myrtales, Sapindales, Caryophyllales, Cornales, Ericales, Lamiales, Solanales [14, 15]. The geography of distribution of naturally transformants is wide, ranging from the tropics and subtropics (neem, representatives of the genus Camellia L.) to forest-tundra and tundra (e.g., representatives of the genus Vaccinium L.), and covers all continents, except for Antarctica [14, 17–20]. The list of natural GMOs includes many cultivated plants used by people from different countries for food as well as medicinal plants. Such plants include tea, guava, peanuts, hops, sweet potatoes, and cranberries. According to preliminary estimates, about 7% of dicotyledonous plants may contain traces of agrobacterial transformation in their genomes. This estimate is based on data on the share of natural GMOs among dicotyledonous species with sequenced genomes [14].

Is it worth searching a single function of cT-DNA for all natural GMOs?

To answer this question, it is necessary to evaluate the cT-DNA structures of natural GMOs and the diversity of intact genes in them.

Before considering the diversity of cT-DNA, we will briefly outline the main groups of genes in it. T-DNA usually contains genes for the synthesis of opines, the products of which are required for the nutrition of bacteria [21]. In addition to opine synthases, cT-DNA encodes oncogenes that cause neoplastic tissue growth and are represented by phytohormone synthesis genes and plast genes. The mechanism of influence of hormonal genes on plant morphogenesis has been known for a long time, while plast genes and the mechanisms of their action have been studied much less [22, 23].

The discussion on the functions of cT-DNA in natural GMOs started from the moment they were described. The discussion was centered on which of the genes (oncogenes or opine synthases) are the most important acquisitions of plants involved in evolution [4]. However, the discussion was hampered by a lack of factual material for research and generalizations. Now the situation has changed. By the beginning of 2021, 36 genera of angiosperms were already known, within which natural transgenic species were described [14, 15]. Among them, the most dominant are those whose cT-DNA contains only genes for the synthesis of opines (Fig. 1, a). This phenomenon can be explained in at least three ways. First, in currently known T-DNA, genes for synthesis of opines are located closer to the right border; therefore, they enter the plant cell first in the course of transformation. If T-DNA transfer is terminated untimely, than the opine synthesis genes could enter the recipient cel without oncogenes [21]. Second, we can expect the presence of strains of agrobacteria whose T-DNA contains only genes for opine synthase. Third, the possibility of transformation of plants with extended T-DNA and the loss of most of it during the evolution of the descendants of the natural transformants with the preservation of only opine synthesis genes cannot be excluded [24].

Fig. 1. Number of genera of natural GMOs with different cT-DNA structures (a) and types of extended cT-DNA structures (b)

Species containing extended T-DNA fragments with oncogenes and genes for opine synthesis rank second in terms of abundance. Extended cT-DNA are mostly represented by inverted imperfect repeats (Fig 1b) and often contain well-known T-DNA genes and new poorly studied sequences, usually attributed to plast genes [14, 15, 25, 26], which indicates a wider variety of agrobacterial strains transforming plants than we previously believed.

The least abundant are cT-DNA containing only oncogenes [14, 15, 24]. Among them, special attention should be paid to plast genes found in representatives of the genera Vaccinium L. and Nyssa Gronov ex L. They are of interest because they are phylogenetically close to the plast gene sequences found in some basidiomycete fungi [14, 15].

Analysis of ORFs in all three types of cT-DNA for the presence of premature stop codons or mutations that cause frame shifts shows that some of the genes there remain intact, while others mutate [14–16]. More intact sequences have been preserved among opine synthases [15] but there are quite a lot of intact ones among plast genes as well.

We can, therefore, conclude that different natural GMOs have different sets of intact genes in their cellular T-DNA. This means that cT-DNA does not have a universal function. In different groups of plants, evolution used forms with various combinations of transgenes, and then far from all of them were preserved intact.

Possible functions of cT-DNA plast genes

Before discussing the cT-DNA plast genes, let us briefly consider what is generally known about this family of genes. The name plast genes comes from the words “developmental plasticity.” The term was coined to emphasize the ability of these genes to change plant morphogenesis in various ways during the transformation of wild-type plants [26]. Levesque et al. [27] suggest that plast genes may have similar functions associated with their common origin, and that their divergence may be an adaptation to different plant species. The best-known members of this family are the genes rolA, rolB, rolC, ORF13, and ORF14 genes from Rhizobium rhizogenes (Riker et al. 1930) Young et al. (2001), gene 6b from Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 (Approved Lists 1980) emend. Hördt et al. 2020 [26]. The analysis of their functions is complex due to the fact that some morphogenetic effects may coincide in different genes, while others may differ. For example, the rolB gene product is sufficient to induce root formation. RolC is not able to independently induce root formation, but exhibits a synergistic effect with rolB [26]. Transgenic plants containing only rolB or only rolC differ in leaf color (the former are dark green, the latter are light green), which indicates antagonism of the action of genes [26]. It is believed that rolB has meristem-inducing activity [28, 29], but it also causes necrosis of tobacco leaves [30] whereas rolC suppresses the necrotic effects induced by rolB. The introduction of rolC into the genome of cultivated tobacco and carnations (Dianthus caryophyllus L.) leads to the stimulation of shoot formation [26, 31], and in the cell culture of ginseng (Panax ginseng C.A. Mey.) it stimulates somatic embryogenesis [32]. The ORF13 gene product enhances the effect of rolB in inducing root generation. However, the expression of orf13 in tobacco [33, 34], tomato [35], and arabidopsis [36] resulted in various changes in the height of transgenic plants up to dwarfism. ORF14 does not have a clear phenotypic effect. However, the fact that the rolA, rolB, rolC, ORF13, ORF14 genes are always located close to one another in T-DNA suggests their possible involvement in the control of the overall process [26]. Possibly, the different combinations of genes from this set function predominantly in different plants. That is, the “inclusion” of specific genes depends on the molecular genetic characteristics of the recipient.

The molecular mechanisms of action of genes remain completely incomprehensible. The fact that plast genes have been described not only in naturally transgenic plants but also in some fungi [22] suggests that if there is a certain common function of all plast genes, it should affect some basic biochemical processes characteristic not only for plants [26].

So far, there has been no direct evidence of the functioning of plast genes of cT-DNA in natural GMOs. However, some indirect arguments clearly indicate that plast genes can affect the growth of natural transformants. The rolC, orf13, and orf14 genes often remain intact in naturally transgenic plants and are expressed [12, 22, 37]. A complete list of expressed genes can be reconstructed from the review [4] and article [14] that we published earlier.

The plast-gene-associated functions of cT-DNA previously discussed in the literature included an increase in root mass, an increase in regenerative capacity, and a transition to earlier flowering and one-year life cycles [13]. There is no clear evidence supporting the latter function. On the contrary, we detected the predominance of perennial forms among nGMOs in the presence of annual nontransgenic species, at least in toadflax [13]. The expanded list of nGMOs for 2021 is interesting in that among the carriers of plast genes, there were many ligneous plant forms, confirming that they are perennial plants [14, 15]. Thus, the new data supports our previous findings. Nevertheless, the identification of new nGMOs with this T-DNA function as a key function cannot be ruled out.

As for the increase in regenerative capacity and induction of root formation, the role of plast genes in the implementation of these functions should be discussed separately. At present, plast gene expression has been demonstrated by molecular methods in representatives of the genera Nicotiana (NgrolC, trolC, Ngorf13, and torf13) [22, 25, 38], Linaria (rolC) [39], Ipomoea (rolB/C-like, orf13, gene c) [10, 40]. When overexpressed in tobacco, Ngorf13 causes the formation of dark green rounded leaves [41], overexpression of NgrolC [42] and trolC [43] leads to a dwarf phenotype and the formation of lanceolate, pale green leaves, whereas torf13 induces green calli on carrot disks [44]. In naturally transgenic toadflax, tissue-specific expression of the LvrolC transgene was demonstrated during plant regeneration from root explants on a hormone-free medium, which may indicate the involvement of the gene product in the regulation of this process [39]. In previous publications, we discussed the effect of rol-genes on the regenerative capacity of Nicotiana species [4, 13]. We concluded that the peculiarities of plant morphogenetic reactions can be determined, among other things, by a combination of intact rol-genes and their level of expression in a particular genotype.

Analysis of the transcriptomes deposited with the NCBI can be used for the primary search for the expressed genes of new natural GMOs. The currently available data can be interpreted in support of expression of plast genes in plants of the genus Vaccinium and Diospyros [14, 15].

The morphogenetic effects of plast genes, their role in the regulation of secondary metabolism and carbohydrate metabolism are discussed in the literature. The most common biologically active secondary plant metabolites include alkaloids, polyphenolic compounds (flavonoids, terpenoids, coumarins, saponins) and essential oils. All these compounds are efficiently synthesized in cultures of hairy roots [45, 46]. Under natural conditions, they are involved in the protection of plants from adverse abiotic factors as well as in the regulation of interaction of plants with other organisms.

Although there is no direct evidence of the involvement of cT-DNA genes in the control of the content of secondary metabolites, this role cannot be ruled out yet. Palazon et al showed that the rolC gene increases nicotine synthesis in transgenic tobacco obtained under laboratory conditions, and the introduction of a cassette from rolA, rolB, and rolC into the plant genome increases the nicotine level more than a single rolC gene does. So, far, no specific mechanisms of regulation of this process have been identified [47–49].

In tobacco, toadflax, and model transgenic arabidopsis plants, the effect of rolC and gene 6b on sugar metabolism was revealed [43, 50]. This function is discussed as some common property of various plast genes, preserved by them from a common ancestral sequence. This hypothesis is confirmed by our preliminary data from the analysis of samples of cranberry (Vaccinium macrocarpon Ait. and V. oxycoccos L.), contrasting with the presence of a full length plast gene previously unknown among agrobacterium strains. The content of monosaccharides is higher in shoots of cranberry forms with a full length plast gene compared to samples containing mutant alleles of the gene with a large deletion [51].

Thus, the involvement of plast genes in the control of plant morphogenesis cannot be ruled out although this process is not known yet. The regulation is probably implemented through the control of carbohydrate metabolism, and perhaps through other mechanisms. The control of plant secondary metabolism is an important component of the regulation of plant-microbial interactions, because secondary metabolites can both attract and repel microbes and insects [45, 46], demonstrating an important ecological role. However, there is another class of compounds that can attract specific microbes. These are opines.

Opine synthase

Opine synthases are the most common transgenes in natural transformants. The functions of opine synthases are quite clear and have been studied in detail in relation to agrobacterial strains of various origins.

In the case of natural GMOs, the greatest success was achieved in the study of opine synthases, for which not only expression at the RNA level was demonstrated in dozens of species [24], but also the ability of plants to accumulate the corresponding opines. Thus, deoxyfructosylglutamine was found in naturally transgenic cultivated tobacco plants, and mikimopine was revealed in the tissues of another nGMO, dodder, [52, 53]. Opines can be used as sources of carbon and nitrogen not only by bacteria, but also by fungi that have enzymes for their catabolism [54]. We can assume that opine synthases play a role in the regulation of the composition of microbial communities in the plant rhizosphere. This is supported by the fact that, in addition to natural GMOs, opine synthase genes have been described in many bacteria outside the group of agrobacteria and are also widely distributed in the genomes of ascomycete fungi [55]. In fungi, a certain mosaic structure was noted in terms of the location on the phylogenetic tree of species and isolates containing specific opine synthases in relation to the owners of other opine synthases within one larger taxon. This aspect can be interpreted in support of the role of horizontal gene transfer in the spread of opine synthases among fungi [55]. Most of the currently discussed opine synthases are annotated in sequenced genomes, based on their homology to known genes, and their real functions are yet to be studied using molecular and biochemical methods. The outcomes of these studies may include already known opines in organisms where they have not been studied before, and new variants of opines that have a certain structural similarity to previously known ones. In addition, new data on the role of opine synthases in the regulation of the formation of plant communities with bacteria and fungi can be expected in the coming years. To confirm this idea, the growing interest in the use of rhizopines to attract beneficial bacteria to the rhizosphere can be indicated. Rhizopines, although different from opines of agrobacteria, perform a similar function to them, attracting rhizobia, capable of metabolizing them, into the plant rhizosphere. In order to control the structure of the microbial community in the laboratory, successful attempts have been made to plants metabolic engineering. The study resulted in transgenic plants synthesizing rhizopines, which, in turn, attracted rhizobia [56]. In the case of natural GMOs, nature has done all the work for us.

If we evaluate the diversity of opine synthases in natural GMOs, then the largest number of intact sequences is described for homologues of mikimopine synthases and cucumopine synthases. Although these are different genes, their products, mikimopine and cucumopine, are isomers [24]. Cucumopine and mikimopine synthases are the most attractive candidates for further research and development of approaches to modify microbial communities in the plant rhizosphere.

Do pGMOs become immune to re-transformation?

To date, species with multiple cT-DNA inserts have been described among natural GMOs. These species belong to the genera such as Nicotiana L., Ipomoea L., Diospyros L., Parasponia Miq., Trema Lour., Silene L. [14, 15, 25]. Among them, special attention should be paid to species of the genera Nicotiana, Diospyros, and Parasponia. Their genomes contain multiple cT-DNA organized as inverted repeats. In Nicotiana tomentosiformis L., all four cT-DNAs in the genome are organized as inverted repeats [25], in Diospyros lotus L., three out of seven are repeats [15], and in Parasponia andersonii Planch. eight out of nine are repeats [14]. Comparing the sequences of the right and left T-DNA arms, we can understand which of them diverged more. Thus, it is possible to perform a relative dating of transformational events (and reveal which of them happened earlier and which one happened later). This simple analysis enables us to draw an important conclusion that the transformational events in the evolution of these genera occurred sequentially, but not simultaneously. When the phylogeny of the genus is studied well by traditional methods, it is possible to link the relative dating to the time scale. Thus, it was revealed that hundreds of thousands of years passed between transformational events in tobacco [25]. Since repeated successive transformations took place in the evolution of representatives of three unrelated plant genera, we can state that the idea of a protective function of cT-DNA against repeated transformations is untenable. On the contrary, it can be assumed that there are species prone to transformation. By identifying their common characteristics and introducing the identified traits into the genomes of other plants, it is possible to optimize further plant transformation protocols in the laboratory.

CONCLUSION

Our review has revealed that horizontal gene transfer from agrobacteria to plants has occurred and is occurring at a higher frequency than previously believed [14]. As a result of such a transfer, forms with various combinations of intact T-DNA genes are preserved in nature, while the other part of the genes mutates or is completely lost. If an extended T-DNA enters the genome during horizontal gene transfer, the forms with cT-DNA organized as an inverted repeat are taken by selection with a higher probability. Probably, this structure suppresses the expression of T-DNA genes, which softens the effect of agrobacterial oncogenes on plant morphogenesis. Further, under conditions of partial suppression of gene expression, selection starts in favor of specific gene combinations. In each case, the combination may be different, which is consistent with Levesque’s idea on divergence of plast genes to different host plants. This results in a wide variety of natural GMOs.

In some plants, the process of such transformation can be repeated several times. It may result in the acquisition of new genes, such as additional opine synthases. Further, cT-DNAs within the same genome can evolve independently of each other.

Recently, evidence is accumulating in support of functioning of T-DNA genes. In the future, we can expect an increase in the number of scientific articles describing new opine synthases, their products, and their functional role in ecosystems.

In addition, the description of new representatives of plast genes can be predicted. This may become valuable material for elucidating the basic functions of the genes of this family.

The accumulated information on new natural GMOs is valuable for future studies of the role of genes obtained by plants from agrobacteria during evolution, for studies of the diversity of agrobacteria strains, and for studies of the function and evolution of cT-DNA genes in natural transformants.

ADDITIONAL INFORMATION

Author contributions. The author confirms that his authorship complies with the international ICMJE criteria.

Funding. This work was supported by the Russian Science Foundation (grant No. 21-14-00050).

About the authors

Tatyana V. Matveeva

Author for correspondence.
Email: radishlet@gmail.com
ORCID iD: 0000-0001-8569-6665
SPIN-code: 3877-6598
Scopus Author ID: 7006494611
ResearcherId: J-6000-2013

Dr. Sci. (Biol.), Professor

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2. Fig. 1. Number of genera of natural GMOs with different cT-DNA structures (a) and types of extended cT-DNA structures (b)

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3. Fig. 1. Number of genera of natural GMOs with different cT-DNA structures (a) and types of extended cT-DNA structures (b)

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