Impact of GBHin the shikimate pathway and direct cascade effects
Despite the widespread use of GBH in agriculture, major questions remain on how its exposure affects cell metabolism and physiology in glyphosate-resistant plants and if there are antagonistic or synergistic effects in stacked-transgene varieties. In order to address these questions, we have profiled transcriptomic changes after GBH treatment and characterized the interactions between the shikimate pathway and other unsupervised metabolic pathways. We were also interested in the direct effects of GBH in native and transgenic EPSPS expression.
Our results showed that native EPSPS expression showed a 1.5 fold change up-regulation in both single-transgene and stacked events, which suggests that reduced levels of AAAs in response to EPSPS inhibition may act as a signal to induce the expression of the shikimate pathway genes and restore the carbon flux through the pathway in plants [37]. In the other hand, insensitive transgenic cp4-epsps showed a decrease in transcript accumulation also in both transgenic varieties. Although stably integrated into the genome, variable and non-directional levels of CP4 EPSPS has been observed linked with other factors, such as the genetic background, trait stacking, growing region or season[38]. But the extent to which detection protocols could differentiate both versions of EPSPS is unclear. We were careful to repeat the full epsps experiment with new seedlings and new herbicide application in order to confirm these results. The underlying mechanism for the decreased cp4-epsps level as well as the role(s) of the native epsps in transgenic varieties remain to be investigated.
While EPSPS enzymes have been equally modulated in both single-transgene and stacked varieties, differences found in downstream metabolism between the varieties suggest synergistic and antagonist effects of GBH when transgenes are stacked. In the stacked variety, the modulation of other amino acids metabolism (cysteine and methionine) and secondary metabolites metabolism, such as flavonoids and glutathione metabolism, as well as the jasmonic acid metabolism, were most prominent (Figure 7). Jasmonic acid derives from fatty acid biosynthesis and increased levels have been also observed after GBH and drought stress application in NK603 herbicide resistant GM maize [20]. GBH was also shown to affect other hormones, such as ethylene [39] and abscisic acid [12].
Intermediates in the shikimate pathway are used for the synthesis of proteins and that in plants also serve as precursors of numerous natural products, such as pigments, alkaloids, hormones, and cell wall components. [40]. Such plant natural compounds play crucial roles in plant growth, development, reproduction, defense, and environmental responses [41]. For example, phenylalanine is a common precursor of numerous phenolic compounds, including lignin, which corresponds to the highest carbon flux [37]. We observed a 3.5 fold change increase of PAL, an enzyme involved in lignin biosynthesis, in the stacked variety. Differently, Zobiole et al [42]. found negative correlation between levels of lignin in single-transgene GM soy and increasing rates of GBH applications. Previous studies also reported higher susceptibility levels to diseases after application of GBH in transgenic varieties which were associated with changes in lignin contend and, consequently, with morphological and functional quality of the plant defense organs [6][43][44].
Toxicity of GBH applications is a known and desired outcome in susceptible plants, whereas in GM tolerant plants, the modulation of shikimate genes is not expected as they contain insensitive and constitutively expressed CP4-EPSPS. Our results show that GM single-transgene and stacked varieties have strong modulation of the shikimate pathway, including EPSPS versions; which also affected numerous compounds derived from shikimate precursor chorismite molecule.
Changes in central carbon metabolism and carbon flux
The constitutive expression of transgenes controlled by strong viral promoters, such as P35S from the cauliflower mosaic virus, has been always a concern due to potential energy cost and carbon flux in plant cells. In this paper, we applied GBH, an inhibitor of the enzyme EPSPS, at recommended concentrations in order to investigate its impact in downstream metabolism in GM tolerant plants containing two or more inserts. Under normal growth conditions, more than 30% of plant-fixed carbon flows through the shikimate pathway [45][46][37]. Whereas under stress, plants mobilize their carbon stocks to transform energy and resist to harmful effects.
In susceptible plants, inhibition of EPSPS reduces the levels of AAAs and their downstream products which act as a signal to induce the expression of the shikimate pathway genes and restore the carbon flux through the pathway. However, the effects of GBH in the central carbon metabolism of GM tolerant plants has been also previously observed.
In our study, we observed a clear difference in carbon metabolism between single-transgene and the stacked variety. While single-transgene variety showed modulation in carbon fixation and glycolysis metabolism, the stacked variety exhibited changes in starch and sucrose metabolism. Our data suggests that single-transgene varieties enhance the cytosolic glycolytic network to provide metabolic flexibility that facilitates plant acclimation to herbicide stress. Modulation of phosphoenolpyruvate carboxykinase that promotes reversible protein phosphorylation of major importance in controlling legume nodule carbon metabolism and related metabolite transport was observed. In addition, other enzymes involved in parallel reactions were also altered; fructose bisphosphate aldolase and malate dehydrogenase genes were also found up regulated. These changes are in agreement with previous studies that have observed changes in GM soybean nodulation after GBH applications [13][47].
In the stacked variety, on the other hand, increased levels of trehalose-phosphate transcripts (3.6 log2FC) were observed. Sucrose and starch balance is directly related to optimization of growth rates [48][49]. Trehalose (α-d-glucopyranosyl-1,1-α-d-glucopyranoside) works as an osmolyte, storage reserve, transport sugar, and stress protectant [50][51]; and it is also involved in growth and development metabolism [52] with clear links to abscisic acid and auxin signaling [53]. Increased levels of trehalose have been observed in response to osmotic stress [54] as well as to dehydration stress tolerance [55] [54]. Most plants accumulate substantial starch reserves in their leaves to provide carbon and energy for maintenance and growth[56][57]. Therefore, the accumulation of soluble sugars, such as trehalose, is suggested to be a protective mechanism under oxidative stress conditions [58][59].
Altered Cellular Redox Homeostasis
Exposure to GBH is directly linked to accumulation of antioxidant enzymes, indicating an oxidative stress [60]. Glutathione (GSH) is a key molecule in the antioxidant network in plants, acting to control reactive oxygen species (ROS) accumulation and facilitating cellular redox homeostasis especially under stress conditions[61]. For instance, GSH plays an important role in herbicide detoxification via the glutathione S-transferase (GST) system [62]. We found evidence for cellular detoxification response through significant up-regulation of GST in both transgenic varieties (single-transgene variety: average log2FC = 3.1; Stacked: average log2FC = 3.5).
On the other hand, other genes encoding important enzymes related to glutathione metabolism showed to be differently affected in the single-transgene and stacked varieties, revealing that both genotypes may respond differently to oxidative stress. For instance, glucose 6-phosphate dehydrogenase (G6PDH) – an enzyme participating in the first two reactions of oxidative pentose phosphate pathway - being significantly down-regulated in the single-transgene variety. Reduced levels of G6PDH is related to glutathione depletion and consequent high oxidative stress in the cell [63]. It is known that reduced glutathione (GSH) is required to combat oxidative stress and maintain the normal reduced state in the cell, a phenomenon known as the redox homeostasis [60][61][16]. Oxidized glutathione (GSSG) is reduced to GSH by NADPH generated by G6PDH in the pentose phosphate pathway [64]. Complete depletion of glutathione in its reduced form (GSH), or the production of GSSG from GSH, with concomitant accumulation of formaldehyde have already been reported as signs of undergoing oxidative stress in single-transgene soybean event as compared to its non-GM isogenic line [19][65].
In the stacked variety, although G6PDH gene expression has not been significantly affected, herbicide treatment up-regulated the expression of 6-phosphogluconate dehydrogenase (6PGDH) gene (log2FC = 1.25). 6PGDH, a second enzyme participating in the OPPP, catalyses the NADP-dependent oxidative decarboxylation of 6-phosphogluconate generating NADPH and ribulose-5-phosphate, a precursor for the synthesis of nucleotides and nucleic acids [66]. We hypothesize that the production of such reducing equivalents is being used in further reductive reactions in stacked plants, such as keeping GSH in its reduced form, aiming at maintaining the cell redox homeostasis.
Our results also showed protein processing in endoplasmatic reticulum (ER) as one of the most up-regulated pathways in both, single-transgene and stacked varieties when GBH is applied. Glutathione homeostasis in response to oxidative stress has been also described as active in the ER [67]. A diverse range of genes encoding important molecular chaperones guiding secretory folding proteins, as well as ubiquitin-proteasomes responsible for exporting and degradation of misfolded proteins, were shown to be significantly up-regulated in the presence of GBH. Since glutathione is oxidized, transport proteins must export GSSG from the ER to the cytosol aiming to reach an ideal glutathione homeostasis [67]. Conversely, the stacked variety showed evidence of oxidative stress responses due to the up-regulation of cytosolic glutathione genes (GST log2FC = 3.5; 6PGDH log2FC = 1.25), while only genes encoding ERAD enzymes were significantly up-regulated in ER. Vivancos et al. [16] have also found effects of herbicide on cellular redox homeostasis of single-transgene, GBH-resistant soybean varieties. More specifically, the authors reported that the accumulation of high levels of glyphosate in GM plants enhanced cellular oxidation, possibly through mechanisms involving increasing of photorespiratory pathway [16]. Moreover, a recent integrative in silico model of C1 metabolism in single-transgene, GBH-resistant GM soybean predicted complete depletion of glutathione and accumulation of formaldehyde as a result of oxidative stress compared to its non-GM counterpart [19]. According to our findings, single-transgene and stacked GM soybean showed oxidative stress at different levels and cellular components.
Photosynthesis imbalance
Photosynthesis efficiency and inhibition of chlorophyll function has been observed as a side-effect from GBH applications in both susceptible and GM tolerant plants [16][68][18]. In other words, GBH seems to impact photosynthesis as a side-effects of glyphosate and its by-products and/or adjuvants whether or not insensitive epsps sequences are present in the genome. In our study, the single-transgene variety showed a decrease in the light-harvesting chlorophyll A and B contend (complex I of class LhcA 2,3 and 4 with four genes involved, and the complex II of class LhcB 1,2,3 and 6 with nine genes involved). These findings are supported by Li et al.[69], which also observed a decline in the content of chlorophyll A and B in GM and conventional soybean varieties under GBH treatment [69]. In addition, we found two genes down-regulated related to putative ferredoxin enzymes. The amount of ferridoxin is also decreased in tobacco under various stresses, including those from herbicide treatment [70]. Iquebal et al. [71]observed that genes involved in the photosynthetic pathway were deregulated after exposure to herbicides in resistant chickpea variety [71]. In Lolium perene sensitive plants, chlorophyll fluorescence was also affected by glyphosate[72].
Defense and environmental responses
Defense imposes a substantial demand for resources that can negatively impact growth and diminish the overall set of energy reserves and/or promote resource diversion for growth, defense, and reduction of photosynthesis [73]. Previous transcriptome studies using microarray technique to investigate the metabolic impact of GBH treatment in susceptible and resistant soybean, arabidopsis and brassica showed that most affected pathways are involved with defense metabolism [15][74][12]. In this study, the single-transgene and stacked varieties showed up regulation of calcium-related pathways, which are essential to coordinate a rapid adaptive response in several species [75][76]. Calmodulin protein families were also altered in both single-transgene variety (two altered genes, average 2.10 log2FC) and stacked (five altered genes, average 3.3 log2FC) varieties. Previous studies with GBH application in sensitive soybean also observed changes in calcium-related genes regardless of herbicide concentrations and the collection time after application (4 and 24 hours)[29][12]. The Ca2+/CaM complex play key roles in plant metabolism as it is the main signal transduction pathway involved in turgor regulation and with an impact in drought tolerance[77].
Rapid recognition of injuries by cellular signal transduction pathways occurs through various signaling molecules, including calcium, protein phosphorylation and ROS, which are well-known triggers of stress resistance in plants [78]. Herbicides are considered abiotic stressors that can disrupt the balance between the production and elimination of ROS [79]. There is a close relationship between calcium-dependent ROS production and a specific group of genes. For example, the respiratory burst oxidase homolog (Rboh) gene family. Activation of this group occurs after the recognition of pathogens and a variety of other processes [80][81]. We observed strong up-regulation of the Rboh group (3.58 log2FC) in the stacked variety. Such oxidases have been reported as key factors in activating innate and mobilized immunity during oxidative stress damage [82].
Another example of defense regulatory circuit was the identification of WRKY transcription factors, which are connected to phosphorylation events of mitogen-activated protein kinase (MAPK) in response to pathogen recognition with the accumulation of Rboh protein [81] Strict regulation and fine-tuning of WRKY proteins are directly linked to plant stress signaling responses [83][84], such as saline stress [85], drought [86][87] and heat stress [88]. We observed up-regulation of WRKY genes in both varieties, with higher expression and number of genes in the stacked variety (five genes with an average of 2.5 fold change).
In addition, pathogenesis-related proteins (PR), known as an indispensable component of innate immune responses in plants under biotic or abiotic stress conditions, were also altered in this study. In the single-transgene variety, we find one gene PR1, up-regulated with a 2.9 log2FC. These proteins are also involved in hypersensitive response or systemic acquired resistance against a variety of plant infections [89] and an important response mechanism to multiple stresses [90]. PR proteins are considered the signature genes of salicylic acid and jasmonic acid pathways in many crop plants [91][92][90][93]. Furthermore, Hsp90 gene families were found up-regulated in the both single-transgene and stacked varieties. In soybean varieties, Hsp90 gene was found induced by heat, salt, and osmotic stresses [94]. Strikingly, the stacked variety up-regulated seven β-glucosidase-related genes with an average of 5 log2FC. We also found a regulated isoflavone 7-O-methyltransferase gene found 8.5 log2FC. a β-glucosidases enable the enzymatic removal of a protecting glucose group, thus providing plants with an immediate chemical defense against protruding herbivores and pathogens[95].
Relevance to risk assessment of stacked GM crops
Worldwide, a growing number of GM crops with stacked transgenic traits are being developed to confer resistance to herbicide active ingredients and some insect species. For most varieties, the single-transgene events might never reach market and pre-market risk assessment. Therefore, an assessment of the risks of the actual GMO to be released in the environment should consider combinatorial and cumulative effects derived from stacking transgenes into single organism.
Omics profiling analysis can contribute to the identification of combinatorial effects that may occur due to interactions among the proteins and metabolites produced by the transgenes or endogenous genes of a stacked GM plant. In addition, interactions between the stacked transgenes or their products, or interactions among the physiological pathways in which the transgenes are involved, taking into account the possibility that these interactions could result in potentially harmful substances, such as anti-nutritional factors, some of which may persist or accumulate in the environment.
Stacked GM plants can be produced through different approaches. In addition to the cross-breeding of two GM plants, multiple traits can be also achieved by the natural cross of transgenic lines that have been found in crop field boundaries [96][97], such as feral transgenic canola outside of cultivation [98][99].
Accordingly, it is reasonable to anticipate future occurrence of stacked traits within ruderal and wild populations. Despite the potential for the formation of feral populations with multiple transgenes, we have little understanding of how these traits could migrate, evolve or influence native and naturalized plant communities. Thus, such profiling studies could generate useful information to assist risk assessment of stacked GM crops and potential feral populations.
Our study on stacked-transgene soybean variety showed GBH effects on shikimate genes, carbon metabolism and flux, photosynthesis, oxidative events and defense response. Whereas GBH effects in single-transgene plants have been reported[100][101][102] our data suggest that GBH affects stacked-transgene plants at a higher extend than its single-transgene near-isogenic comparator. In addition, GBH adverse effects in GM tolerant plants are widely variable depending on species and cultivar and herbicide regime. Therefore, it is intrinsically important to elucidate the GBH effects on physiological processes related to metabolic disturbances in order to better understand the glyphosate-herbicidal mechanism and its possible unintended effects on commercialized transgenic varieties.