Differential regulation of a MYB transcription factor is correlated with transgenerational epigenetic inheritance of trichome density in Mimulus guttatus

Epigenetic inheritance, transgenerational transmission of traits not proximally determined by DNA sequence, has been linked to transmission of chromatin modifications and gene regulation, which are known to be sensitive to environmental factors. Mimulus guttatus increases trichome (plant hair) density in response to simulated herbivore damage. Increased density is expressed in progeny even if progeny do not experience damage. To better understand epigenetic inheritance of trichome production, we tested the hypothesis that candidate gene expression states are inherited in response to parental damage. Using M. guttatus recombinant inbred lines, offspring of leaf-damaged and control plants were raised without damage. Relative expression of candidate trichome development genes was measured in offspring. Line and parental damage effects on trichome density were measured. Associations between gene expression, trichome density, and response to parental damage were determined. We identified M. guttatus MYB MIXTA-like 8 as a possible negative regulator of trichome development. We found that parental leaf damage induces down-regulation of MYB MIXTA-like 8 in progeny, which is associated with epigenetically inherited increased trichome density. Our results link epigenetic transmission of an ecologically important trait with differential gene expression states – providing insight into a mechanism underlying environmentally induced ‘soft inheritance’. Keywords: maternal effects, Mimulus guttatus (yellow monkeyflower), MYB transcription factors, phenotypic plasticity, soft inheritance, transgenerational epigenetic inheritance, trichome density, wound response Introduction Heritable variation is usually attributed to DNA sequence variants that are stable, causative, and derived from random mutations (Lynch & Walsh, 1998). However, epigenetic inheritance has now been shown in over a hundred different cases involving bacteria, protists, fungi, plants, and animals (reviewed in Jablonka & Raz, 2009). Epigenetic inheritance, also referred to as cross-generational phenotypic plasticity, is the transmission of phenotypes that are not proximally determined by DNA. In the narrowest sense, epigenetic inheritance results from the transfer of modified chromatin states (e.g. DNA methylation, histone modification, and associations with nonhistone proteins) through the gametes of sexually reproducing organisms (Lippman et al., 2003, 2004; Rangwala et al., 2006; Rassoulzadegan et al., 2006; Richards, 2006; Chandler, 2007; Jablonka & Raz, 2009; Daxinger & Whitelaw, 2010; Richards et al., 2010). In a broader sense, epigenetic inheritance also includes maternal environmental effects (Rossiter, 1996; Jablonka & Lamb, 2008). As our understanding of mechanisms underlying both narrow-sense epigenetic inheritance and the transmission of maternal effects expands, the line between the two becomes quite blurred (Richards et al., 2010). For instance, maternal environment may result in transmission of small molecules, including small RNAs, to progeny, which in turn, may function to establish or maintain chromatin modifications (Matzke & Birchler, 2005). Both definitions of epigenetic inheritance will include phenomena that affect phenotypes across generations by altering gene expression profiles. A growing number of studies have demonstrated epigenetic inheritance of environmentally induced phenotypes (Agrawal et al., 1999; Anway et al., 2005; Benyshek et al., 2006; Cropley et al., 2006; Crews et al., 2007). This so-called ‘soft inheritance’, in which the environment can alter a malleable hereditary material, is fundamentally different from DNA-based inheritance (Mayr, 1980, 1982; Richards, 2006). Mechanisms of ‘soft inheritance’ may evolve when environmental conditions experienced by parents and offspring are correlated and it is advantageous to provide information about the environment across generations (Bossdorf et al., 2008). Mimulus guttatus (yellow monkeyflower) exhibits epigenetic inheritance of trichome density, a putative defense trait, in response to leaf damage (Holeski, 2007). Trichomes are plant hairs that serve a variety of defensive and physiological functions. In M. guttatus they are straight, unicellular, mostly glandular, and often secrete a sticky and potentially noxious fluid. Leaf trichomes have been shown to reduce insect herbivory in a number of species (Levin, 1973; Agren & Schemske, 1993; Mauricio, 1998; Romeis et al., 1999). They interfere with insect movement and feeding, and the fluids they secrete may trap, poison, or repel herbivores (Elle & Hare, 2000). Constitutive production of trichomes is variable both within and among populations of M. guttatus, and simulated insect damage to leaves induces a within-generation increase in the production of trichomes. This induced state can be transmitted to offspring and expressed even if those offspring do not experience damage (Holeski, 2007). Trichomes are a model trait for the study of plant cellular differentiation, development, and patterning (Szymanski et al., 2000; Larkin et al., 2003; Hulskamp, 2004; Serna & Martin, 2006; Ishida et al., 2008; Zhao et al., 2008). Consequently, there is detailed information on genes and developmental pathways involved in trichome development from two distantly related core eudicots, Antirrhinum majus (asterid) and Arabidopsis thaliana (rosid). In Antirrhinum, four closely related paralogous transcription factors, MIXTA, MYB MIXTA LIKE 1 (AmMYBML1), AmMYBML2, and AmMYBML3, have been implicated in the promotion of trichome development. Overexpression of these genes leads to increased trichome density, as well as trichome development from epidermal cells that would otherwise not produce trichomes (Glover et al., 1998; Perez-Rodriguez et al., 2005; Baumann et al., 2007; Jaffe et al., 2007). In addition, MIXTA-like homologs from Arabidopsis and cotton have been shown to be both negative and positive regulators of trichome differentiation (Jakoby et al., 2008; Machado et al., 2009; Gilding & Marks, 2010). MIXTA-like genes therefore appear to affect trichome development across major core eudicot lineages. An alternate pathway has been identified in Arabidopsis, where a known transcriptional activation/repression complex consisting of multiple proteins likely acts as a regulator of GLABRA2 (GL2) expression, and GL2, when activated, promotes trichome development (Rerie et al., 1994; Szymanski et al., 1998, 2000; Fyvie et al., 2000; Ohashi et al., 2002; Schiefelbein, 2003; Pesch & Hulskamp, 2004; Serna, 2004; Ishida et al., 2007, 2008; Simon et al., 2007; Wang et al., 2007; Wang & Chen, 2008; Zhao et al., 2008). Interestingly, this GL2-dependent trichome initiation pathway appears to be conserved in nonArabidopsis rosid species, including cotton (Suo et al., 2003; Wang et al., 2004; Humphries et al., 2005; Guan et al., 2008), but appears to be absent from Antirrhinum and other asterid species (Payne et al., 1999; Ramsay & Glover, 2005; Serna & Martin, 2006). In this study, we examine associations between epigenetic inheritance of damage-induced trichome production and expression of candidate trichome development genes in M. guttatus (asterid). We begin by identifying M. guttatus orthologs of MIXTA-like genes and GL2. Because of their widely conserved role in regulating epidermal outgrowth, including trichome differentiation, MIXTA-like homologs are excellent candidate genes for identifying underlying developmental genetic pathways involved in trichome production and differentiation in M. guttatus. In addition, if a GL2-dependent pathway functions to initiate trichome development in M. guttatus, any differences in the expression of upstream regulators of the M. guttatus GL2 homolog will be reflected in the relative expression levels of GL2 itself. Therefore, GL2, because of its downstream position in the Arabidopsis trichome development pathway, is a good candidate gene for assessing the effect of this putative pathway on trichome induction in M. guttatus. Here we identify eight MIXTA-like homologs and a single GL2 ortholog in M. guttatus. We measure the associations between trichome density, parental damage, and expression of MIXTA-like and GL2 candidate genes using a panel of recombinant inbred lines (RILs) developed from two disparate natural populations of M. guttatus. This panel exhibits an average increase in trichome density of undamaged offspring when parental plants experience damage, that is, epigenetic inheritance of induction, genetic variation in epigenetic inheritance of induction (Holeski, 2007), and variation in constitutive trichome production. By measuring six different M. guttatus lines (four RILs and the two parental lines from which they were derived), we evaluate interactions between genetic and epigenetic variation at the phenotypic level, and compare associations between epigenetic variation, genetic variation, and gene expression. This represents a system in which the basis of environmentally induced, heritable epigenetic variation can be studied in conjunction with the effect of genetic variation in an ecologically relevant trait.