Mechanisms of transgenerational responses

Plastic responses to environmental stress can be transmitted across plant generations via multiple mechanisms, independently or even in the absence of DNA sequence variation (Cortijo et al., 2014; Zhang et al., 2013). In particular, adaptive transgenerational plasticity can be mediated by starch reserves, mRNAs, proteins, hormones, and other primary and secondary metabolites packaged in the seed (Roach and Wulff, 1987; Leishman et al., 2000; Fenner and Thompson, 2005; Moles and Leishman, 2008), and by epigenetic mechanisms (Rossiter, 1996; Boyko et al., 2010; Richards et al., 2017), via the alteration of gene expression through heritable changes in cytosine methylation or histone modification (Richards, 2006; Bird, 2007; Richards et al., 2017). Since provisioning effects are mediated directly by maternal individuals, environmental effects that persist for multiple generations must be mediated by mechanisms capable of longer-term stability (e.g. epigenetic mechanisms). However, these mechanisms are neither completely separate nor mutually exclusive. On the contrary, multiple mechanisms can cooperatively influence heritable phenotypes (see paragraph: "Combined effects of transgenerational plasticity mechanisms").

a. Seed provisioning and maternally derived proteins and mRNAs

Seed provisioning refers to the carbohydrate, lipid, protein, and mineral nutrient reserves stored by the mother plant in the developing seed (Koller, 1972; Srivastava, 2002). It is often reduced in the deprivation of resources in maternal plants, resulting in an offspring with diminished early growth rates, seedling size, and competitive ability (Haig and Westoby, 1988; Fenner and Thompson, 2005), causing maladaptive transgenerational effects. Alternatively, resource-deprived maternal plants can maintain or even increase seed provisioning (Roach and Wulff, 1987; Schmitt et al., 1992; Sultan, 1996, 2001; Donohue and Schmitt, 1998), maximizing in this way seedling survival. For example, well-provisioned offspring can produce more extensive root systems in dry soil or larger shoot systems under canopy shade (Silvertown, 1984; Wulff, 1986; Leishman et al., 2000). In natural populations, however, the adaptive benefit of such enhanced provisioning may be limited. Resource-deprived maternal plants, in such cases, tend to produce fewer seeds with more chances of surviving. Furthermore, an increased seed provisioning can correlate with decreased persistence in the soil seed bank (Sultan, 1996; Donohue and Schmitt, 1998; Fenner and Thompson, 2005). Hence, in stressful maternal conditions, transgenerational effects mediated via seed provisioning can promote offspring success in specific ecological settings.

Besides seed provisioning, stressed maternal plants can transmit to the offspring also proteins, mRNAs, small RNAs, secondary metabolites, and hormones. Maternally-derived proteins can act both as regulatory molecules and as nutritive elements (via seed provisioning). Together with maternally-derived mRNAs, they are key regulators of seed dormancy and germination (Donohue, 2009). Since stress can significantly alter maternal gene expression, maternally-derived mRNAs and proteins may facilitate adaptive growth responses in seeds germinating under stressful conditions (Rajjou et al., 2004).

b. Epigenetic inheritance: DNA methylation, histone modifications and small RNAs

Epigenetic mechanisms can also mediate transgenerational effects. DNA methylation seems to be both environmentally sensitive and heritable over multiple (i.e., ≥8) generations (Johannes et al., 2009; Reinders et al., 2009; Hauser et al., 2011; reviewed by Jablonka and Raz, 2009). Therefore, DNA methylation (and possibly other epigenetic mechanisms) may also play a role in regulating transgenerational effects of environmental stress (Kalisz and Purugganan, 2004; Grant-Downton and Dickinson, 2006; Boyko and Kovalchuk, 2011). After exposing A. thaliana plants to salt stress, Boyko et al. (2010) found an increased tolerance to the same stress in the progeny that correlated with the inheritance of stress-induced DNA methylation marks. In tobacco plants, infection with tobacco mosaic virus (TMV) also caused heritable changes in DNA methylation. It increased resistance to viral, bacterial, and fungal pathogens in the progeny (Kathiria et al., 2010).

To some degree, the transgenerational effects mediated by DNA methylation seem to be genotype-specific (Herman and Sultan, 2016; Rendina González et al., 2018). Herman and Sultan (2016) investigated the transmission mechanisms of adaptive transgenerational responses to drought stress in different genetic lines of the annual plant Polygonum persicaria. The offspring of the drought-stressed parental plants were treated with the demethylating agent zebularine and grown in dry soil. These plants did not present the adaptive phenotypes shown by the naturally methylated offspring (more extended root systems and greater biomass), suggesting that demethylation removed the adaptive effect of parental drought stress (without significantly altering phenotypic expression in offspring of well-watered parents). Since the seed provisioning between offspring of drought-stressed and well-watered parents was equivalent, differential seed provisioning did not contribute to the effect of parental drought on offspring phenotypes. Furthermore, the effect of demethylation on the expression of the parental drought effect was found to differ among the genetic lines. These results suggest that DNA methylation can mediate adaptive, genotype-specific effects of parental stress on offspring phenotypes. However, demethylation of the whole genome is not targeted and may result in random demethylation variation among replicate lines (see also “Chapter 3: Plant defense response”) or may activate previously inactive transposable elements (see also “Chapter 4: Epigenetics in evolution”).

Despite being to some extent genotype-specific, plastic transgenerational responses to environmental stress can occur even in the absence of genetic variation. To investigate this aspect, a potent approach is the use of A. thaliana epigenetic recombinant inbred lines (epiRILs), characterized by high DNA methylation variation but no DNA sequence variation (Johannes et al., 2009; Reinders et al., 2009; Teixeira et al., 2009). In A. thaliana, studies of epiRILs showed that DNA methylation variants can cause substantial heritable variation in key traits such as primary root length and flowering time (Cortijo et al., 2014; Zhang et al., 2013). In particular, Zhang et al. (2013) tested the response of a large number of epiRILs of A. thaliana to drought and increased nutrient conditions, and they found significant variance components and heritabilities in several phenotypic traits, including flowering time, plant height and total biomass, fruit number, and root:shoot ratio. Thus, this study provides evidence that variation in DNA methylation can cause substantial heritable variation of ecologically important plant traits even in the absence of genetic variation.

In sexually reproducing plants, another layer of complexity is represented by the parental sex, since the inheritance of epigenetic modifications mediating transgenerational effects seems to be sex-specific. In the yellow monkeyflower plants (Mimulus guttatus), Akkerman et al. (2016) tested for differences between maternal and paternal transmission of the transgenerational induction of increased glandular trichome density in response to simulated insect damage. Both maternal and paternal damage resulted in similar and additive increases in progeny trichome production. Notably, the treatment of germinating seeds with 5-azaC erased the effect of maternal but not paternal damage. These results indicate that transgenerational effects can occur through maternal and paternal germlines, but they differ in the proximate mechanism of epigenetic inheritance.

Not only DNA methylation, but even histone modifications can affect gene expression by altering chromatin structure. Histone modifications can also be transferred from one generation to the other, as shown in A. thaliana (Lang-Mladek et al., 2010). Lang-Mladek et al. (2010) found that both heat stress and UV-B exposure could induce heritable changes in gene expression in A. thaliana, correlating with histone H3 deacetylation and with no DNA methylation changes. This effect was found only in small groups of cells within the plant and persisted for two offspring generations.

In addition to DNA methylation and histone modifications, small RNAs (sRNAs) can also have a role in plant transgenerational effects. Changes in sRNA composition have been associated with heat (Ito et al., 2011; Bilichak et al., 2015; Song et al., 2016), drought (Matsui et al., 2008; Tricker et al., 2012), salinity (Borsani et al., 2005; Matsui et al., 2008; Ding et al., 2009; Song et al., 2016), cold, and osmotic stress (Song et al., 2016), and in some cases they even persisted in the offspring of stressed plants (Bilichak et al., 2015; Morgado et al., 2017). In A. thaliana, mutants in the biogenesis of sRNA showed compromised transgenerational caterpillar herbivore resistance (Rasmann et al., 2012), suggesting that sRNAs were required to sustain induced defense responses across generations.

c. Combined effects of transgenerational plasticity mechanisms

As mentioned previously, several modes of transgenerational responses can act together to influence offspring phenotypes. In the wild radish Raphanus raphanistrum, both seed mass-dependent and seed mass-independent transgenerational responses to maternal caterpillar herbivory were found (Agrawal et al., 1999; Agrawal, 2001, 2002). Seed mass is often used as a proxy for seed provisioning (Moles and Leishman, 2008, Lacey et al., 1997). The molecular mechanism behind non-provisioning effects was not investigated in this study (Agrawal, 2001, 2002). However, these results show how even a relatively simple environmental change can induce multiple physiological changes that together enhance offspring performance.

d. Transgenerational plasticity in clonally reproducing plants

Transgenerational effects have been studied mostly across sexual generations, but they have high potential relevance, especially for asexual (or clonal) species (e.g. Latzel and Klimešová, 2010). When plants reproduce clonally, they produce genetically identical offspring not arising from seeds (except in the case of apomixis, in which seeds are asexually produced). Therefore, seed provisioning cannot play a role in transgenerational effects occurring in non-apomictic clonal plants. On the contrary, epigenetic inheritance can occur both across clonal and sexual reproduction (reviewed by Jablonka and Raz, 2009). Epigenetic inheritance even seems to be more prominent across clones, which bypass the epigenetic erasure associated with meiosis (Feng et al., 2010). Thus, it has been suggested that epigenetic inheritance could play a key role in transgenerational plasticity in clonally reproducing plants (Latzel and Klimešová, 2010; Verhoeven and Preite, 2014; González et al., 2016; Rendina González et al., 2018).

González et al. (2016) exposed plants of white clover (Trifolium repens) to different drought treatments and analyzed the transgenerational effects (i.e. offspring biomass) on the untreated clonal offspring. To assess whether DNA methylation was essential to mediate these effects, half of the plants were treated with the demethylating agent 5-azacitidine. In the naturally methylated clonal offspring, they found stress-driven transgenerational effects. However, these effects were not present in the demethylated plants, suggesting that DNA methylation was involved in the observed transgenerational effects.

In the same system, Rendina González et al., 2018 explored whether the effects of transgenerational plasticity were genotype-specific and under epigenetic control. They analyzed the effects of transgenerational plasticity induced by multiple parental stresses on the clonal offspring using five different genotypes. They found that transgenerational plasticity induced by different stresses was genotype-specific and that at least one stress (drought) induced DNA methylation variation that was maintained across several clonal offspring generations. These results suggest that transgenerational effects in Trifolium repens are genotype-specific, potentially under epigenetic control and inherited across several clonal generations.