Beyond Transcript Concentrations: Quantifying Polyploid Expression Responses per Biomass, per Genome, and per Cell with RNA-Seq.

RNA-seq has been used extensively to study expression responses to polyploidy. Most current methods for normalizing RNA-seq data yield estimates of transcript concentrations (transcripts per transcriptome). The implicit assumption of these normalization methods is that transcriptome size is equivalent between the samples being compared such that transcript concentrations are equivalent to transcripts per cell. In recent years, however, evidence has mounted that transcriptome size can vary dramatically in response to a range of factors including polyploidy and that such variation is ubiquitous. Where such variation exists, transcript concentration is often a poor or even misleading proxy for expression responses at other biologically relevant scales (e.g., expression per cell). Thus, it is important that transcriptomic studies of polyploids move beyond simply comparing transcript concentrations if we are to gain a complete understanding of how genome multiplication affects gene expression. I discuss this issue in more detail and summarize a suite of approaches that can leverage RNA-seq to quantify expression responses per genome, per cell, and per unit of biomass.

Polyploidy and microbiome associations mediate similar responses to pathogens in Arabidopsis.

It has become increasingly clear that the microbiome plays a critical role in shaping the host organism's response to disease. There also exists mounting evidence that an organism's ploidy level is important in their response to pathogens and parasites. However, no study has determined whether or how these two factors influence one another. We investigate the effect of whole-genome duplication in Arabidopsis thaliana on the above-ground (phyllosphere) microbiome and determine the interacting impacts of ploidy and microbiome on disease outcome. Using seven independently derived synthetic autotetraploid Arabidopsis accessions and a synthetic leaf-associated bacterial community, we confirm that polyploids are generally more resistant to the model pathogen Pseudomonas syringae pv. Tomato DC3000. Polyploids fare better against the pathogen than diploids do, regardless of microbial inoculation, whereas diploids harboring an intact microbiome have lower pathogen densities than those without. In addition, diploids have elevated numbers of defense-related genes that are differentially expressed in the presence of their phyllosphere microbiota, whereas polyploids exhibit some constitutively activated defenses, regardless of colonization by the synthetic community. These results imply that whole-genome duplication can enhance immunity, resulting in a decreased dependence on the microbiome for protection against pathogens.

Fifteen compelling open questions in plant cell biology.

As scientists, we are at least as excited about the open questions-the things we do not know-as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

Estimating somatic mutation rates by bottlenecked duplex sequencing in non-model organisms: Daphnia magna as a case study.

Somatic mutations are evolutionarily important as determinants of individual organismal fitness, as well as being a focus of clinical research on age-related disease, such as cancer. Identifying somatic mutations and quantifying mutation rates, however, is extremely challenging and genome-wide somatic mutation rates have only been reported for a few model organisms. Here, we describe the application of Duplex Sequencing on bottlenecked WGS libraries to quantify somatic nuclear genome-wide base substitution rates in Daphnia magna. Daphnia, historically an ecological model system, has more recently been the focus of mutation studies, in part because of its high germline mutation rates. Using our protocol and pipeline, we estimate a somatic mutation rate of 5.6 × 10-7 substitutions per site (in a genotype where the germline rate is 3.60 × 10-9 substitutions per site per generation). To obtain this estimate, we tested multiple dilution levels to maximize sequencing efficiency and developed bioinformatic filters needed to minimize false positives when a high-quality reference genome is not available. In addition to laying the groundwork for estimating genotypic variation in rates of somatic mutations within D. magna, we provide a framework for quantifying somatic mutations in other non-model systems, and also highlight recent innovations to single molecule sequencing that will help to further refine such estimates.

Expression Partitioning of Duplicate Genes at Single Cell Resolution in Arabidopsis Roots.

Gene duplication is a key evolutionary phenomenon, prevalent in all organisms but particularly so in plants, where whole genome duplication (WGD; polyploidy) is a major force in genome evolution. Much effort has been expended in attempting to understand the evolution of duplicate genes, addressing such questions as why some paralog pairs rapidly return to single copy status whereas, in other pairs, both paralogs are retained and may diverge in expression pattern or function. The effect of a gene - its site of expression and thus the initial locus of its function - occurs at the level of a cell comprising a single cell type at a given state of the cell's development. Using Arabidopsis thaliana single cell transcriptomic data we categorized patterns of expression for 11,470 duplicate gene pairs across 36 cell clusters comprising nine cell types and their developmental states. Among these 11,470 pairs, 10,187 (88.8%) had at least one copy expressed in at least one of the 36 cell clusters. Pairs produced by WGD more often had both paralogs expressed in root cells than did pairs produced by small scale duplications. Three quarters of gene pairs expressed in the 36 cell clusters (7,608/10,187) showed extreme expression bias in at least one cluster, including 352 cases of reciprocal bias, a pattern consistent with expression subfunctionalization. More than twice as many pairs showed reciprocal expression bias between cell states than between cell types or between roots and leaves. A group of 33 gene pairs with reciprocal expression bias showed evidence of concerted divergence of gene networks in stele vs. epidermis. Pairs with both paralogs expressed without bias were less likely to have paralogs with divergent mutant phenotypes; such bias-free pairs showed evidence of preservation by maintenance of dosage balance. Overall, we found considerable evidence of shifts in gene expression following duplication, including in >80% of pairs encoding 7,653 genes expressed ubiquitously in all root cell types and states for which we inferred the polarity of change.

Robust Cytonuclear Coordination of Transcription in Nascent Arabidopsis thaliana Autopolyploids.

Polyploidy is hypothesized to cause dosage imbalances between the nucleus and the other genome-containing organelles (mitochondria and plastids), but the evidence for this is limited. We performed RNA-seq on Arabidopsis thaliana diploids and their derived autopolyploids to quantify the degree of inter-genome coordination of transcriptional responses to nuclear whole genome duplication in two different organs (sepals and rosette leaves). We show that nuclear and organellar genomes exhibit highly coordinated responses in both organs. First, organelle genome copy number increased in response to nuclear whole genome duplication (WGD), at least partially compensating for altered nuclear genome dosage. Second, transcriptional output of the different cellular compartments is tuned to maintain diploid-like levels of relative expression among interacting genes. In particular, plastid genes and nuclear genes whose products are plastid-targeted show coordinated down-regulation, such that their expression levels relative to each other remain constant across ploidy levels. Conversely, mitochondrial genes and nuclear genes with mitochondrial targeting show either constant or coordinated up-regulation of expression relative to other nuclear genes. Thus, cytonuclear coordination is robust to changes in nuclear ploidy level, with diploid-like balance in transcript abundances achieved within three generations after nuclear whole genome duplication.

Expression-level support for gene dosage sensitivity in three Glycine subgenus Glycine polyploids and their diploid progenitors.

Retention or loss of paralogs following duplication correlates strongly with the function of the gene and whether the gene was duplicated by whole-genome duplication (WGD) or by small-scale duplication. Selection on relative gene dosage (to maintain proper stoichiometry among interacting proteins) has been invoked to explain these patterns of duplicate gene retention and loss. In order for gene dosage to be visible to natural selection, there must necessarily be a correlation between gene copy number and gene expression level (transcript abundance), but this has rarely been examined. We used RNA-Seq data from seven Glycine subgenus Glycine species (three recently formed allotetraploids and their four diploid progenitors) to determine if expression patterns and gene dosage responses at the level of transcription are consistent with selection on relative gene dosage. As predicted, metabolic pathways and gene ontologies that are putatively dosage-sensitive based on duplication history exhibited reduced expression variance across species, and more coordinated expression responses to recent WGD, relative to putatively dosage-insensitive networks. We conclude that selection on relative dosage has played an important role in shaping gene networks in Glycine.

Variation in transcriptome size: are we getting the message?

The number of RNA molecules per cell (transcriptome size) is highly variable, differing among and within cell types depending on cell size, stage of the cell cycle, ploidy level, age, disease state, and growth condition. Such variation has been observed at the level of total RNA, ribosomal RNA, messenger RNA (mRNA), and the polyadenylated fraction of mRNA, and these distinct RNA species can also vary in abundance with respect to each other. This variation in transcriptome size has been largely ignored or overlooked, and in fact, standard data normalization procedures for transcript profiling experiments implicitly assume that mRNA transcriptome size is constant. Consequently, variation in transcriptome size has important technical implications for such experiments, as well as profound biological implications for the affected cells and underlying genomes. Here, we review what is known about transcriptome size variation, explore how ignoring this variation introduces systematic bias into standard expression profiling experiments, and present examples of how such biases have led to erroneous conclusions in expression studies of sex chromosome dosage compensation, cancer, Rett syndrome, embryonic development, aging, and polyploidy. We also discuss how quantifying transcriptome size will help to elucidate the selective forces underlying patterns of gene and genome evolution and review the evidence that cells exert tight control over transcriptome size in order to maintain cell size homeostasis and to optimize chemical reactions within the cell, such that loss of control over transcriptome size is associated with cancer and aging. Thus, transcriptome size is an important phenotype in its own right. Finally, we discuss strategies for quantifying transcriptome size and individual gene dosage responses in order to account for and better understand this important biological phenomenon.

The wild side of a major crop: soybean's perennial cousins from Down Under.

The accumulation of over 30 years of basic research on the biology, genetic variation, and evolution of the wild perennial relatives of soybean (Glycine max) provides a foundation to improve cultivated soybean. The cultivated soybean and its wild progenitor, G. soja, have a center of origin in eastern Asia and are the only two species in the annual subgenus Soja. Systematic and evolutionary studies of the ca. 30 perennial species of subgenus Glycine, native to Australia, have benefited from the availability of the G. max genomic sequence. The perennial species harbor many traits of interest to soybean breeders, among them resistance to major soybean pathogens such as cyst nematode and leaf rust. New species in the Australian subgenus continue to be described, due to the collection of new material and to insights gleaned through systematic studies of accessions in germplasm collections. Ongoing studies in perennial species focus on genomic regions that contain genes for key traits relevant to soybean breeding. These comparisons also include the homoeologous regions that are the result of polyploidy in the common ancestor of all Glycine species. Subgenus Glycine includes a complex of recently formed allopolyploids that are the focus of studies aimed at elucidating genomic, transcriptomic, physiological, taxonomic, morphological, developmental, and ecological processes related to polyploid evolution. Here we review what has been learned over the past 30 years and outline ongoing work on photosynthesis, nitrogen fixation, and floral biology, much of it drawing on new technologies and resources.

Mining transcriptomic data to study the origins and evolution of a plant allopolyploid complex.

Allopolyploidy combines two progenitor genomes in the same nucleus. It is a common speciation process, especially in plants. Deciphering the origins of polyploid species is a complex problem due to, among other things, extinct progenitors, multiple origins, gene flow between different polyploid populations, and loss of parental contributions through gene or chromosome loss. Among the perennial species of Glycine, the plant genus that includes the cultivated soybean (G. max), are eight allopolyploid species, three of which are studied here. Previous crossing studies and molecular systematic results from two nuclear gene sequences led to hypotheses of origin for these species from among extant diploid species. We use several phylogenetic and population genomics approaches to clarify the origins of the genomes of three of these allopolyploid species using single nucleotide polymorphism data and a guided transcriptome assembly. The results support the hypothesis that all three polyploid species are fixed hybrids combining the genomes of the two putative parents hypothesized on the basis of previous work. Based on mapping to the soybean reference genome, there appear to be no large regions for which one homoeologous contribution is missing. Phylogenetic analyses of 27 selected transcripts using a coalescent approach also are consistent with multiple origins for these allopolyploid species, and suggest that origins occurred within the last several hundred thousand years.

The legacy of diploid progenitors in allopolyploid gene expression patterns.

Allopolyploidization (hybridization and whole-genome duplication) is a common phenomenon in plant evolution with immediate saltational effects on genome structure and gene expression. New technologies have allowed rapid progress over the past decade in our understanding of the consequences of allopolyploidy. A major question, raised by early pioneer of this field Leslie Gottlieb, concerned the extent to which gene expression differences among duplicate genes present in an allopolyploid are a legacy of expression differences that were already present in the progenitor diploid species. Addressing this question necessitates phylogenetically well-understood natural study systems, appropriate technology, availability of genomic resources and a suitable analytical framework, including a sufficiently detailed and generally accepted terminology. Here, we review these requirements and illustrate their application to a natural study system that Gottlieb worked on and recommended for this purpose: recent allopolyploids of Tragopogon (Asteraceae). We reanalyse recent data from this system within the conceptual framework of parental legacies on duplicate gene expression in allopolyploids. On a broader level, we highlight the intellectual connection between Gottlieb's phrasing of this issue and the more contemporary framework of cis- versus trans-regulation of duplicate gene expression in allopolyploid plants.

Extensive translational regulation of gene expression in an allopolyploid (Glycine dolichocarpa).

All flowering plants have experienced repeated rounds of polyploidy (whole-genome duplication), which has in turn driven the evolution of novel phenotypes and ecological tolerances and been a major driver of speciation. The effects of polyploidy on gene expression have been studied extensively at the level of transcription and, to a much lesser extent, at the level of the steady state proteome, but not at the level of translation. We used polysome profiling by RNA-Seq to quantify translational regulation of gene expression in a recently formed (∼100,000 years ago) allotetraploid (Glycine dolichocarpa) closely related to the cultivated soybean (Glycine max). We show that there is a high level of concordance between the allopolyploid transcriptome and translatome overall but that at least one-quarter of the transcriptome is translationally regulated. We further show that translational regulation preferentially targets genes involved in transcription, translation, and photosynthesis, causes regional and possibly whole-chromosome shifts in expression bias between duplicated genes (homoeologs), and reduces transcriptional differences between the polyploid and its diploid progenitors, possibly attenuating misregulation resulting from genome merger and/or doubling. Finally, translational regulation correlates positively with long-term retention of homoeologs from a paleopolyploidy event, suggesting that it plays a significant role in polyploid evolution.

A comparative transcriptomic study of an allotetraploid and its diploid progenitors illustrates the unique advantages and challenges of RNA-seq in plant species.

PREMISE OF THE STUDY: RNA-seq analysis of plant transcriptomes poses unique challenges due to the highly duplicated nature of plant genomes. We address these challenges in the context of recently formed polyploid species and detail an RNA-seq experiment comparing the leaf transcriptome profile of an allopolyploid relative of soybean with the diploid species that contributed its homoeologous genomes. METHODS: RNA-seq reads were obtained from the three species and were aligned against the genome sequence of Glycine max. Transcript levels were estimated for each gene, relative contributions of polyploidy-duplicated loci (homoeologues) in the tetraploid were identified, and comparisons of transcript profiles and individual genes were used to analyze the regulation of transcript levels. KEY RESULTS: We present a novel metric developed to address issues arising from high degrees of gene space duplication and a method for dissecting a gene's measured transcript level in a polyploid species into the relative contribution of its homoeologues. We identify the gene family likely contributing to differences in photosynthetic rate between the allotetraploid and its progenitors and show that the tetraploid appears to be using the "redundant" gene copies in novel ways. CONCLUSIONS: Given the prevalence of polyploidy events in plants, we believe many of the approaches developed here to be applicable, and often necessary, in most plant RNA-seq experiments. The deep sampling provided by RNA-seq allows us to dissect the genetic underpinnings of specific phenotypes as well as examine complex interactions within polyploid genomes.

Anatomical, biochemical, and photosynthetic responses to recent allopolyploidy in Glycine dolichocarpa (Fabaceae).

PREMISE OF THE STUDY: Previous studies have shown that polyploidy has pronounced effects on photosynthesis. Most of these studies have focused on synthetic or recently formed autopolyploids, and comparatively little is known about the integrated effects of natural allopolyploidy, which involves hybridity and genome doubling and often incorporates multiple genotypes through recurrent origins and lineage recombination. METHODS: Glycine dolichocarpa (designated T2) is a natural allotetraploid with multiple origins. We quantified 21 anatomical, biochemical, and physiological phenotypes relating to photosynthesis in T2 and its diploid progenitors, G. tomentella (D3) and G. syndetika (D4). To assess how direction of cross affects these phenotypes, we included three T2 accessions having D3-like plastids (T2(D3)) and two accessions having D4-like plastids (T2(D4)). KEY RESULTS: T2 accessions were transgressive (more extreme than any diploid accession) for 17 of 21 phenotypes, and species means differed significantly in T2 vs. both progenitors for four of 21 phenotypes (higher for guard cell length, electron transport capacity [J(max)] per palisade cell, and J(max) per mesophyll cell; lower for palisade cells per unit leaf area). Within T2, four of 21 parameters differed significantly between T2(D3) and T2(D4) (palisade cell volume; chloroplast number and volume per unit leaf area; and J(max) per unit leaf area). CONCLUSIONS: T2 is characterized by transgressive photosynthesis-related phenotypes (including an ca. 2-fold increase in J(max) per cell), as well as by significant intraspecies variation correlating with plastid type. These data indicate prominent roles for both nucleotypic effects and cytoplasmic factors in photosynthetic responses to allopolyploidy.

Comparative evolution of photosynthetic genes in response to polyploid and nonpolyploid duplication.

The likelihood of duplicate gene retention following polyploidy varies by functional properties (e.g. gene ontologies or protein family domains), but little is known about the effects of whole-genome duplication on gene networks related by a common physiological process. Here, we examined the effects of both polyploid and nonpolyploid duplications on genes encoding the major functional groups of photosynthesis (photosystem I, photosystem II, the light-harvesting complex, and the Calvin cycle) in the cultivated soybean (Glycine max), which has experienced two rounds of whole-genome duplication. Photosystem gene families exhibit retention patterns consistent with dosage sensitivity (preferential retention of polyploid duplicates and elimination of nonpolyploid duplicates), whereas Calvin cycle and light-harvesting complex gene families do not. We observed similar patterns in barrel medic (Medicago truncatula), which shared the older genome duplication with soybean but has evolved independently for approximately 50 million years, and in Arabidopsis (Arabidopsis thaliana), which experienced two nested polyploidy events independent from the legume duplications. In both soybean and Arabidopsis, Calvin cycle gene duplicates exhibit a greater capacity for functional differentiation than do duplicates within the photosystems, which likely explains the greater retention of ancient, nonpolyploid duplicates and larger average gene family size for the Calvin cycle relative to the photosystems.

Quantifying whole transcriptome size, a prerequisite for understanding transcriptome evolution across species: an example from a plant allopolyploid.

Evolutionary biologists are increasingly comparing gene expression patterns across species. Due to the way in which expression assays are normalized, such studies provide no direct information about expression per gene copy (dosage responses) or per cell and can give a misleading picture of genes that are differentially expressed. We describe an assay for estimating relative expression per cell. When used in conjunction with transcript profiling data, it is possible to compare the sizes of whole transcriptomes, which in turn makes it possible to compare expression per cell for each gene in the transcript profiling data set. We applied this approach, using quantitative reverse transcriptase-polymerase chain reaction and high throughput RNA sequencing, to a recently formed allopolyploid and showed that its leaf transcriptome was approximately 1.4-fold larger than either progenitor transcriptome (70% of the sum of the progenitor transcriptomes). In contrast, the allopolyploid genome is 94.3% as large as the sum of its progenitor genomes and retains > or =93.5% of the sum of its progenitor gene complements. Thus, "transcriptome downsizing" is greater than genome downsizing. Using this transcriptome size estimate, we inferred dosage responses for several thousand genes and showed that the majority exhibit partial dosage compensation. Homoeologue silencing is nonrandomly distributed across dosage responses, with genes showing extreme responses in either direction significantly more likely to have a silent homoeologue. This experimental approach will add value to transcript profiling experiments involving interspecies and interploidy comparisons by converting expression per transcriptome to expression per genome, eliminating the need for assumptions about transcriptome size.