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Epigenetic mechanisms, such as DNA methylation, can cause stable alterations in gene activity without changes in the underlying DNA sequence. DNA methylation is associated with silencing of transposons, imprinting and silencing of both transgenes and endogenous genes (Kooter et al., 1999; Grossniklaus et al., 2001; Miura et al., 2001; Lippman et al., 2004; Shiba et al., 2006; Zilberman et al., 2007). In mammals, resetting of DNA methylation takes place during early embryonic development (Santos et al., 2002). In plants, by contrast, a considerable proportion of DNA methylation marks can be stably transmitted from parents to offspring (Kakutani et al., 1999; Vaughn et al., 2007; Johannes et al., 2009), and many examples exist of methylation epi-alleles that cause segregating phenotypes (Cubas et al., 1999; Kalisz & Purugganan, 2004; Richards, 2006).
The combination of heritability and phenotypic consequences of DNA methylation suggests that the mechanism could play a role in natural selection and adaptation, in ways that may not be explained by DNA sequence variation (Rapp & Wendel, 2005; Grant-Downton & Dickinson, 2006; Richards, 2006; Bossdorf et al., 2008; Boyko & Kovalchuk, 2008; Jablonka & Raz, 2009). In order to evaluate an evolutionary role of epigenetic inheritance, it is important to first gain a better insight into the processes that generate methylation variation between individuals. These processes are currently poorly understood. Although microarray and bisulfite sequencing studies provide a detailed but static picture of the genomic methylation landscape in plants (Cokus et al., 2008; Lister et al., 2008; Zhang, 2008), it largely remains to be determined how responsive the methylation code is to internal and external cues.
Major genomic events, such as hybridization and polyploidization (Adams & Wendel, 2005; Dong et al., 2006; Chen, 2007; Paun et al., 2007), and also environmental stresses (Chinnusamy & Zhu, 2009), can trigger DNA methylation changes in plants. Stress-induced methylation changes may be targeted specifically to stress-related genes. Alternatively, methylation changes may generate nonspecific (random) differences between individuals, which may have adaptive significance during times of stress (Rapp & Wendel, 2005), because they increase the range of variation that natural selection can act upon. Whether stress-targeted or random, environmentally induced methylation variation may add an interesting epigenetic component to population responses to natural selection. It is therefore relevant to establish whether environment-induced methylation modification is a common phenomenon, and whether induced methylation changes are stably transmitted to next generations.
The evolutionary relevance of epigenetic variation requires that it is not simply a direct downstream consequence of genetic (DNA sequence) variation. Only when epigenetic and genetic variation are independent, or at least not fully dependent, can epigenetic inheritance affect evolutionary processes in ways that cannot be explained by sequence variation (Richards, 2006, 2008; Bossdorf et al., 2008). Unraveling epigenetic from genetic variation can be a difficult task in genetically diverse populations (Johannes et al., 2008). This is one of the main obstacles to evaluating the evolutionary relevance of epigenetic inheritance. However, detecting independent epigenetic variation is considerably less complicated in populations that lack genetic variation (Johannes et al., 2009; Verhoeven et al., in press). In this study, we explored stress-induced methylation variation in apomictic dandelion plants. Apomictic dandelions reproduce through unfertilized seeds, and offspring are genetic copies of the mother plant (van Dijk, 2003). The dandelion system thus has the advantage that epigenetic alterations can be studied in the absence of genetic variation.
The system is also interesting to study from an evolutionary perspective. The evolutionary potential of apomictic lineages is severely limited because of the absence of genetic variation that is normally associated with sexual reproduction. There are indications that apomictic dandelions may have compensatory mechanisms to generate heritable variation, for instance via increased transposon activity or somatic recombination (Richards, 1989; King & Schaal, 1990). A similar enhanced role for epigenetic variation might be hypothesized.
In this study, we exploited the genetic identity of apomictic clone members by exposing the same genotype to different environments and evaluating the methylation consequences in stressed plants and in their unstressed offspring. This can detect stress-induced and heritable epigenetic variation that does not directly reflect genetic variation. Using methylation-sensitive amplified fragment length polymorphisms (MS-AFLPs) to assess methylation variation at genome-wide, anonymous marker loci, we specifically asked: do salt stress, nutrient stress and chemical induction of anti-herbivore and anti-pathogen defenses promote methylation changes? And, if so, are these changes transmitted to offspring?
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Our results show that environmental stresses readily induce DNA methylation changes at a genome-wide scale and demonstrate that most of the induced changes are faithfully transmitted to offspring. Because we used an apomictic study species, the observed methylation variation in the offspring of stressed plants is associated with parental environments and not with genetic variation among plants. Thus, the results reflect transgenerational epigenetic plasticity of a single genotype in response to environmental stress. We found some evidence that specific stresses can trigger specific methylation changes, leading, for instance, to epigenetic divergence between control and salicylic acid-treated plants. However, there was also general evidence for a stress-induced increase in methylation variation within treatment groups, especially in generation 2, with replicate plants showing limited consistency in their methylation changes. Thus, ecological stresses promote autonomous, heritable epigenetic variation and, depending on phenotypic effects, this variation is available for natural selection to act upon. With genetic inheritance, the role of the environment in evolutionary processes is essentially to select among heritable variation but, with epigenetic inheritance, the environment may have an additional role of generating heritable variation at the moment at which it is most required, i.e. during times of stress.
Stress effects on methylation patterns were statistically detectable, even in this relatively small experiment, but it was not typically observed that individual loci showed a consistent methylation change as a result of stress (shared among the majority of replicates), whereas control plants remained unchanged. Rather, there was a subset of inherently unstable loci that were often also polymorphic within the control group, and the effect of stresses was to increase the likelihood that methylation changes occurred at these loci. The functional interpretation of this pattern is unclear. Consistent methylation changes that are controlled by, for instance, hormone signals may occur at specific genes (Kim et al., 2009), but highly localized and specific responses will not generally be captured by AFLPs. In order to gain a better insight into the functional significance of stress-induced methylation changes, it will be important to evaluate the sequence context of AFLPs and, more generally, to take a gene-level approach to the evaluation of stress effects on methylation patterns.
There has been considerable speculation about the evolutionary implications of stress-induced epigenetic variation (e.g. Rapp & Wendel, 2005; Grant-Downton & Dickinson, 2006; Richards, 2006; Bossdorf et al., 2008; Boyko & Kovalchuk, 2008; Jablonka & Raz, 2009). However, current evidence in plants that ecological stresses induce methylation repatterning is limited to a few examples, and the heritability of such induced methylation changes has remained largely unknown. Using methylation-sensitive AFLPs, methylation changes at anonymous marker loci have been reported previously in response to heavy metal stress in hemp and clover (mostly hypomethylations; see Aina et al., 2004), water deficit stress in pea roots (mostly hypermethylations; Labra et al., 2002) and viral infection in tomato (Mason et al., 2008). In tobacco, viral infection (Wada et al., 2004) and several abiotic stresses (Choi & Sano, 2007) caused demethylation and the associated upregulated expression of stress-related genes. A rare example of transgenerational methylation effects has been documented in tobacco, in which virus-infected plants produced offspring with globally hypermethylated genomes, but with hypomethylated defense-related R loci (Boyko et al., 2007). Our results show that environment-induced methylation changes, when they occur, are generally transmitted faithfully to the next generation.
The independence between genetic and epigenetic variation is a key feature of our experiment. However, it is likely that some low-level genetic variation is generated by stresses, via stress-induced transposon activity (Capy et al., 2000; Slotkin & Martienssen, 2007) or increased somatic recombination rates (Molinier et al., 2006). Both processes can, in fact, result from stress-induced demethylation, because DNA methylation functions to suppress transposons (Miura et al., 2001) and might also shield genomic regions from somatic recombination (Boyko et al., 2007). It is therefore possible that some MS-AFLP variation in our experiment is associated with induced genetic variation. However, if such genetic modifications occurred, they were clearly not sufficiently pronounced to cause detectable AFLP variation, and thus it seems unlikely that induced genetic variation is responsible for the large MS-AFLP variation that was observed.
Within a single apomictic dandelion genotype, as in our experiment, the observed epigenetic variation is not associated with genetic variation. In natural populations that consist of multiple apomictic lineages, however, there could very well be a genetic component to environmentally induced epigenetic plasticity, because different genotypes may express different levels of plasticity. This is often observed with transgenerational phenotypic plasticity (Sultan, 1996; Galloway, 2001; Holeski, 2007). Genotypes with higher propensity to methylation alterations will show higher within-genotype epigenetic variability. In such situations, the relationship between genetic and epigenetic polymorphisms may be weak, if stress-induced methylation changes are random rather than targeted to specific loci. We detected stress-induced random methylation changes, but only within a subset of susceptible loci. The majority of MS-AFLP loci remained unaffected across generations and treatments. Variation in methylation stability between loci may result from differences in the underlying mechanisms that generate and maintain DNA methylation in different genomic contexts (Chan et al., 2005). Some regions, notably some transposable elements and other repeats, are under the control of RNAi-guided DNA methylation, and these regions remain very stably methylated, but methylation in other contexts can be less strictly controlled (Richards, 2006; Henderson & Jacobsen, 2007; Teixeira et al., 2009).
In comparing the number of methylation changes per experimental group, we found strong statistical evidence that more incidences of methylation change occurred in several treatment groups than in the control group. However, some individuals showed higher overall propensity to express methylation changes than others, and only the SA–control comparison stood up to a more conservative test that accounts for the fact that markers scored in the same individual are not independent. In this analysis, all other comparisons at best approached the subsignificance level in generation 2. Therefore, a cautious overall interpretation of these statistical results is that it is very probable that treatments other than SA trigger heritable methylation changes; however, this awaits confirmation in follow-up studies.
Our study demonstrates the fundamental point that ecological stresses cause autonomous DNA methylation variation, at a genome-wide scale, that is transmitted to offspring. This highlights the potential of epigenetic inheritance to play a role in evolutionary processes. However, important questions remain to be addressed. First, our study does not provide an insight into the stability of stress-induced methylation changes beyond the first generation. Methylation changes that are caused by 5-azacytidine demethylation or by mutants that are deficient in methylation enzymes are often stable for many generations (Stokes et al., 2002; Fieldes et al., 2005; Akimoto et al., 2007; Johannes et al., 2009), but this remains to be demonstrated for changes that are induced by ecological stresses. Second, and importantly, the phenotypic consequences of the observed methylation changes are unknown. In our experiment, we observed that offspring traits were significantly affected by each of the four stress treatments, and these transgenerational effects were not simply attributable to treatment-related differences in seed biomass (KJF Verhoeven, unpublished). In some cases, maternal stress exposure enhanced the offspring responses when exposed to the same stress. For instance, all plants responded to nutrient stress by increasing the root : shoot biomass ratio (this allocation to root tissue is a well-known strategy to capture more of the limiting nutrient resources; Gedroc et al., 1996), but the increase was significantly larger in offspring of nutrient-stressed mothers relative to the offspring of control mothers. Such seemingly adaptive transgenerational effects can persist for multiple generations (Whittle et al., 2009) and our data are certainly consistent with an underlying epigenetic mechanism for this phenomenon. However, from our current data, it cannot be established whether the observed methylation effects and observed phenotypic effects are in fact causally related.
Apomictic reproduction in Taraxacum involves the production of unreduced egg cells via a modified process of female meiosis that circumvents normal meiotic I reductional division, and is further characterized by embryo and endosperm development without fertilization (Vijverberg & van Dijk, 2007). It is possible that these deviations from normal sexual reproduction affect the inheritance of methylation patterns, as epigenetic regulation may take place during these phases in sexual reproduction (Santos et al., 2002; Slotkin et al., 2009). It is currently unknown whether or not apomictic reproduction is unusually permissive to transgenerational inheritance of methylation marks, and thus whether our observations are specific to asexual reproduction or are more general. This issue could be addressed in future studies that also include sexual T. officinale, which co-occur with apomictic conspecifics in nature (van Dijk, 2003).
Striking heritable phenotypic variation is sometimes observed in the absence of detectable genetic variation, and epigenetic variation is a candidate mechanism to account for such observations (Richards et al., 2008). Our work demonstrates that heritable DNA methylation variation is readily generated in apomictic dandelions. Depending on long-term stability and phenotypic effects, such variation might add to the heritable plasticity and to the evolutionary potential of apomictic lineages that have limited genetic variation.