Drought is a major abiotic stress that limits crop productivity . Climate change models predict an increase in summer drying in the midlatitudes, which could contribute to an increase in the number of episodes of drought [2, 3]. Engineering plants with enhanced tolerance of abiotic stresses such as drought is a major objective of plant biotechnology that is expected to be commercialized in the near future [4, 5]. Tolerance to abiotic stress may be achieved through the modification of endogenous plant pathways, often by manipulating important regulatory proteins such as transcription factors. Altering the level of expression of key transcription factors involved in abiotic stress pathways has been shown to enhance tolerance to various abiotic stresses in Arabidopsis [6–9] as well as in important crop species such as rice [10–12], maize , and alfalfa .
Traits involving tolerance to abiotic stresses are considered to be more complex than those that are currently commercialized due to the large number of genes and pathways that may be affected. Furthermore, the interaction between plants and the environment is an intricate, continuous process that has been difficult to characterize, further adding to the complexity of manipulating abiotic stress tolerance traits. The increased complexity of these traits may correspond with a greater potential for unintended effects to occur in transgenic plants.
In transgenic systems, two different types of unintended effects are generally known to occur . Position effects are attributed to the insertion of a transgene at a particular locus in the genome and the resulting interference this might cause. These effects will vary with the site of integration and will therefore be unique to each independent plant line. Position effects can be easily eliminated by screening for plant lines that have no or little position effects. In contrast, pleiotropic effects are independent of the site of transgene insertion and are the sum of all the phenotypic effects caused by expression of the transgene. While some of these may be the intended trait, others may occur through unexpected interactions of the gene with plant processes and constitute the unintended pleiotropic effects. These effects are of greater interest since they are more difficult to eliminate and more likely to create safety issues.
Engineering more complex traits such as abiotic stress tolerance in plants through the manipulation of transcription factors may uncover cryptic properties of the transcription factor that could produce some of the unintended pleiotropic effects. Many transcription factors are part of large families that have complex evolutionary histories [16, 17]. These families typically arise through gene duplications followed by functional divergence in separate expression domains or through the acquisition of new functions. These processes often result in functional redundancies within the families that can be difficult to detect. Furthermore, some transcription factors may retain ancestral functions that are sometimes only revealed by altering the normal pattern of expression. Therefore the manipulation of transcription factors in engineering complex traits such as abiotic stress tolerance may be likely to produce unintended pleiotropic effects.
The use of non-targeted global profiling technologies, such as microarray analysis, to identify unintended effects in plant systems has proven an effective means of determining the "substantial equivalence" of a transgenic plant to its non-transgenic counterpart. Such approaches have been used to investigate unintended effects in a number of transgenic plant systems [18–27]. To date, these studies have primarily focused on simple, monogenic traits such as those that are currently commercially grown. As transgenic crops with more complex traits involving the modification of endogenous plant pathways will soon be entering the market, it is important to extend these analyses to investigate the potential for unintended pleiotropic effects in such systems.
In order to understand the extent and kinds of unintended effects that could be induced in transgenic plants engineered for complex traits, we conferred drought tolerance on Arabidopsis thaliana by overexpressing the transcription factor ABF3. This system targets drought resistance, a trait that will likely enter the market in the near future. Since transcription factors ultimately function by altering the levels of expression of target genes, we investigated unintended effects using microarray analysis to survey global gene expression profiles. In order to eliminate position effects in our analysis and focus on the pleiotropic unintended effects, we employed the Cre/l ox system to excise the ABF3 transgene from the site of insertion, leaving behind the selectable marker, to create control plant lines. Without the ABF3 transgene, the pleiotropic effects will be absent but the site of integration is still interrupted by the selectable marker such that position effects are maintained in these lines.
ABF3 belongs to the ABF/AREB subfamily of bZIP transcription factors which consists of thirteen members in Arabidopsis. Several members have been shown to function in ABA signalling either during seed maturation or in response to stress . These factors can bind to ABA-response elements (ABREs), cis -regulatory elements found in the promoters of many ABA- and stress-responsive genes [29–31]. In addition to drought tolerance, overexpression of ABF3 confers tolerance to salt, cold, heat, and oxidative stresses, suggesting that it regulates multiple abiotic stress pathways in Arabidopsis [7, 32]. Three other ABF/AREB transcription factors are predicted to function in ABA-dependent stress signalling based on expression profiling and overexpression studies. Expression of ABF1 is induced by cold-treatment . ABF2/AREB1 is induced by salt-treatment as well as dehydration but not cold and overexpression confers tolerance to a wide range of abiotic stresses, including salt, drought, heat, and oxidative stress [29, 31, 33]. Interestingly, ABF2/AREB1 also appears to function in glucose signalling as well as in the regulation of seedling growth . ABF4/AREB2 is expressed in response to cold, drought, and salt and overexpression renders plants tolerant to drought and salt [7, 29, 31]. Therefore, ABF3 likely shares some redundant functions with other members of the ABF/AREB subfamily.
Plant response to drought involves changes in the expression patterns of a large number of genes [34–36] and, in addition to members of the ABF/AREB family, a number of other transcription factors have been identified that play a role in the drought response in Arabidopsis. These include the AP2/ERF transcription factors DREB2A, DREB2B, and CBF4 [8, 37, 38], AtMYB2, which functions in concert with AtMYC2 [39–41], and the NAC family transcription factors ANAC19, ANAC055/ATNAC3, and ANAC072/RD26 , which at least partially function in concert with a zinc finger homeodomain protein ZFHD1 . Therefore, while overexpression of ABF3 affects one of the key drought response pathways, it is not the only pathway mediating the drought response at the gene expression level.
The use of the Cre/lox system to create control lines also created an opportunity to examine the effects of the Cre/l ox system on the transcriptome. Site-specific recombination technologies can be used to excise selectable markers or other undesirable genetic elements and can also be used to direct site-specific integration of transgenes . While many studies have employed Cre-mediated recombination in plant systems with no apparent unintended effects [45–48], other studies have observed a range of abnormal phenotypes including growth defects, leaf chlorosis, delayed flowering, and male sterility [49, 50]. In tobacco plants transformed with a chloroplast targeted Cre recombinase, recombination was observed involving cryptic lox sites in the plastid genome, but invariably the second lox site was located within the transgene [51–53]. These recombinations could result in deletions of up to 147 kb, but they did not cause any deleterious effects in the plants [51–53]. Studies in animal systems have similarly revealed that Cre recombinase can have unintended effects, often leading to chromosomal aberrations [54–57].
These studies suggest that the Cre/lox system has the potential to cause unintended effects in plant systems, by mediating recombination with cryptic lox sites that may be present in the genome resulting in large deletions. Such cryptic lox sites are difficult to identify since they may deviate substantially from conventional loxP sites  and not all of the unintended effects may produce readily apparent phenotypic abnormalities, so studying the unintended effects of Cre recombinase using a non-targeted approach such as microarray analysis is essential for establishing the utility and safety of this technology.
In this study, we performed microarray analysis on Arabidopsis plants engineered to be drought-tolerant through overexpression of the transcription factor ABF3 with the goal of identifying unintended pleiotropic effects. The results suggest that overexpression of ABF3 has a minimal impact on the transcriptome, with differences in the gene expression pattern only detectable in response to drought and then being suggestive of transcriptional reprogramming as opposed to the activation of novel pathways. In addition, we examined the impact of Cre recombinase on the transcriptome to detect any unintended effects of this technology and found that it had minimal effects on gene expression patterns in plants following transgene excision.