Autopolyploidy: an epigenetic macromutation

Abstract

Polyploidy—whole-genome duplication—is a key process in plant evolution, with one or more duplication events evident in the genomes of nearly all land plant lineages (Leebens-Mack et al., 2019). Although polyploidization often also involves hybridization (allopolyploidy), it is clear that doubling the genome, in and of itself (autopolyploidy), is a macromutation: a single event with many phenotypic consequences. The mechanisms by which this macromutation alters phenotypes remain mysterious, particularly at the cellular level (Doyle and Coate, 2019), but at least some of them fall under the broad heading of “epigenetics”, which Cavalli and Heard (2019, p. 489) define as “molecules and mechanisms that can perpetuate alternative gene activity states in the context of the same DNA sequence”. Doubling a diploid genome creates an autotetraploid that typically will differ in gene expression and phenotype from its isogenic diploid progenitor (e.g., Robinson et al., 2018; Corneillie et al., 2019) and can also differ in classic epigenetic features such as methylation pattern (Zhang et al., 2015). Such an autopolyploid thus has qualitatively “the same DNA sequence” as its diploid progenitor yet has “alternate gene activity states” that it can “perpetuate” over many generations. In short, autopolyploidy is, in and of itself, an epigenetic macromutation. Accordingly, techniques for studying the epigenome have begun to shed new light on what autopolyploidy “does”, affording insights into the direct effects of genome doubling minus the dramatic impact of hybridity that characterizes allopolyploids (e.g., Wendel et al., 2018), and which also do not involve millions of years of adaptation to changes in genome size (e.g., Roddy et al., 2020). An autopolyploid that is isogenic with its diploid progenitor differs from it quantitatively, in doubled dosage of every gene and in doubled DNA content. Both effects lead to phenotypic changes not only through altered gene expression but also by directly or indirectly increasing the sizes of cells and organelles, leading to quantitative changes such as decreased surface to volume ratios (e.g., of the nucleus or plastid), increased distances molecules must travel (e.g., mRNAs from a larger nucleus to the cytoplasm), and altered concentrations (e.g., of transcription factors in the nucleus). Transcriptional dosage responses to experimentally doubling the genome of Arabidopsis thaliana are nonlinear and variable among genes (Fig. 1A). There is often a global reduction in expression on a per-genome basis, combined with differential effects on the regulation of particular classes of genes, notably those that are dosage sensitive (Hou et al., 2018; Coate et al., 2020; Song et al., 2020). Neither overall patterns of methylation nor proximity of transposable elements to genes appears to explain these genome-wide or gene-class-specific phenomena, leaving chromatin folding in space and time (the 4D nucleome; Dekker et al., 2017) as a possible mechanism for coordinated responses of numerous unlinked genes. Hou et al. (2018) suggested that deviation from doubling of the overall mRNA transcriptome in autotetraploid A. thaliana could be a result of increased (but not doubled) cell size. Implication of cell size in global control of transcription brings up the “nucleotype” hypothesis: that cell size and associated phenotypes (e.g., nuclear volume, cell cycle duration) are strongly influenced by bulk DNA content rather than being determined entirely by genotype, perhaps as a consequence of biophysical laws (Bennett, 1971; Doyle and Coate, 2019). What is the relationship between the nucleotype and the epigenome? Altered metabolism is one possibility, not only due to its effects on all aspects of cell biology, including chromatin dynamics (Sharma and Rando, 2017), but also given the interdependence of cell size and metabolic activity, and the impact of gene dosage on metabolic flux (Bekaert et al., 2011). Altered dimensions also affect crowding and compaction, which in the case of chromatin is known to affect gene expression. New insights into the epigenetics of genome doubling have been provided by a high-throughput chromosome conformation capture (Hi-C) study comparing chromatin organization of a synthetic A. thaliana autopolyploid with its Col-0 diploid progenitor (Zhang et al., 2019; Fig. 1B–F). In the doubled nucleus, individual duplicated chromosomes appear to maintain separate chromosome territories. Nuclear volume increases with genome size (Simova and Herben, 2012), but genome doubling produces less than a doubling in nuclear volume (Sas-Nowosielska and Bernas, 2016; Robinson et al., 2018), which could affect chromatin compaction and alter contacts among chromosomes. Indeed, Zhang et al. (2019) found increased inter-chromosomal interactions and reduced intra-chromosome arm interactions in the autotetraploid. Moreover, about 12% of the genome, including over 2600 genes, changed position between loose vs. condensed structural domains (LSD vs. CSD), which correspond, respectively, to the largely euchromatic, transcriptionally active “A” and heterochromatic, transcriptionally repressed “B” compartments found in animals and many plants. Previously, such alteration of partitioning in a polyploid had been described in a natural allopolyploid (Wang et al., 2018), but there the effect of genome doubling is complicated by hybridity and evolutionary divergence. In the synthetic A. thaliana autopolyploid, there was evidence of increased compaction and greater repressive methylation in CSDs, and looser structure and increased activating methylation in LSDs, but overall histone methylation patterns were not altered dramatically. Zhang et al. (2019) reported that nearly 750 genes were differentially expressed between the diploid and autotetraploid, many of them involved in stress responses as in other autopolyploids (Coate and Doyle, 2019). Differential expression did not correlate with changed methylation pattern, but over 70% of genes with altered expression were located in regions of the genome that showed changes in their levels of cis- and trans-interactions; in particular, promoter–promoter interaction frequency was higher in the autotetraploid. In contrast to this strong effect of interaction, fewer than 30 of the 2600 genes in genomic regions that shifted between CSDs and LSDs were differentially expressed, suggesting that chromatin interaction could be a more important determinant of gene expression shifts than overall chromatin compaction when the genome doubles. Among the differentially expressed genes was the flowering time repressor, FLC, whose increased expression correlated with altered cis-chromatin interactions in the later-flowering autopolyploid. The involvement of a gene known to be affected by cell size (Ietswaart et al., 2017) suggests general links among altered chromatin, gene expression, and novel functional traits produced by genome multiplication. Importantly, this example concerns a phenotype that could certainly be acted upon by natural selection, thus emphasizing the evolutionary relevance of the epigenome in autopolyploidy. Additional studies that integrate epigenomics, nucleome architecture, gene expression, and phenomics will ultimately be necessary to understand fully the effects and consequences of genome multiplication and whether there are “rules” that dictate responses to polyploidization. For one thing, the Zhang et al. (2019) study used tissue from aerial parts of whole seedlings, and as interesting as their results are, they represent the average effect of genome doubling on many different cell types. But not all cell types respond to genome duplication the same way—or at all. Robinson et al. (2018) found that although both epidermal pavement cells and stomatal guard cells increased in area with increased ploidy, the two cell types had different response curves. Even more dramatically, Katagiri et al. (2016) found that although leaf epidermal pavement cells were larger in synthetic A. thaliana autopolyploids, palisade mesophyll cells were not. Moreover, when Katagiri et al. (2016) induced palisade cells to express ATML1, a transcription factor whose ectopic expression generates epidermal features, the transformed palisade cells behaved like epidermal cells and enlarged in response to polyploidization. Consequently, any rules governing cell size and other phenotypic responses to polyploidy are likely specific to a particular cell type. It is now possible to study plant cell biology at single-cell resolution, not only transcriptomically, but with Hi-C (Zhou et al., 2019), and we look forward to the application of these approaches to synthetic autopolyploids. Identifying the connections between phenotypes and chromatin-level variation is a key step toward mechanistic understanding of any epigenetic phenomenon, but it is only a first step. Separating cause from effect is hindered by the observation that the same phenotype can be produced either genetically, generally by altering gene function, or epigenetically, by mis-regulation due to alteration of chromatin context (Zoghbi and Beaudet, 2016). Moreover, mutations in genes directly involved in epigenetic processes (e.g., chromatin remodelers) can cause phenotypes by altering the epigenetic states at one or more downstream loci. Zoghbi and Beaudet (2016, p. 11) conclude that all of these effects “are not caused by epigenetic mutations, but the mutated genes secondarily alter chromatin states that are critical components of the epigenotype”. We suggest that whole organism autopolyploids could be a useful model for dissecting the effects of epigenetic modifications of phenotypes because in autopolyploids the alteration of chromatin is not the direct result of a specific genetic mutation. Rather, even though gene action could certainly be involved through feedbacks involving metabolic changes caused by nucleotypic and/or gene dosage effects (e.g., increased cell size, organelle number; Fig. 1) it is clear that, in an autopolyploid, genome duplication has a direct effect on chromatin state. In the Zhang et al. (2019) study, each gene whose transcription level or chromatin compartment is altered in the autopolyploid thus represents an opportunity to explore the interrelations between chromatin changes and gene expression in a genetic background unaltered by conventional genetic mutations. In conclusion, whole-genome duplication causes sweeping alterations to the 4D nucleome, which likely drive phenotypic changes independent of classic genetic mutation, making autopolyploidy an epigenomic macromutation. Emerging techniques to quantify chromatin-level changes will yield key insights into the effects of “pure polyploidy” (Spoelhof et al., 2017) and position autopolyploids as key models for understanding epigenetic interactions and their effects on evolutionarily relevant phenotypes. We thank Pam Diggle for encouragement and for comments on several versions of the manuscript. We are grateful to our two reviewers; Adam Roddy was particularly helpful. Jonathan Wendel provided numerous insightful comments and suggestions that improved the final version of the manuscript. We apologize to all of the authors whose work we could not cite due to the limited number of references allowed. The authors’ work on polyploidy has been supported by the U.S. National Science Foundation, most recently by award 1257522. J.J.D. and J.E.C. contributed equally both conceptually and in writing the manuscript.

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