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.

Whole-Genome Sequence of Synthesized Allopolyploids in Cucumis Reveals Insights into the Genome Evolution of Allopolyploidization.

The importance of allopolyploidy in plant evolution has been widely recognized. The genetic changes triggered by allopolyploidy, however, are not yet fully understood due to inconsistent phenomena reported across diverse species. The construction of synthetic polyploids offers a controlled approach to systematically reveal genomic changes that occur during the process of polyploidy. This study reports the first fully sequenced synthetic allopolyploid constructed from a cross between Cucumis sativus and C. hystrix, with high-quality assembly. The two subgenomes are confidently partitioned and the C. sativus-originated subgenome predominates over the C. hystrix-originated subgenome, retaining more sequences and showing higher homeologous gene expression. Most of the genomic changes emerge immediately after interspecific hybridization. Analysis of a series of genome sequences from several generations (S0, S4-S13) of C. ×hytivus confirms that genomic changes occurred in the very first generations, subsequently slowing down as the process of diploidization is initiated. The duplicated genome of the allopolyploid with double genes from both parents broadens the genetic base of C. ×hytivus, resulting in enhanced phenotypic plasticity. This study provides novel insights into plant polyploid genome evolution and demonstrates a promising strategy for the development of a wide array of novel plant species and varieties through artificial polyploidization.

Genome evolution in Oryza allopolyploids of various ages: Insights into the process of diploidization.

The prevalence and recurrence of whole-genome duplication in plants and its major role in evolution have been well recognized. Despite great efforts, many aspects of genome evolution, particularly the temporal progression of genomic responses to allopolyploidy and the underlying mechanisms, remain poorly understood. The rice genus Oryza consists of both recently formed and older allopolyploid species, representing an attractive system for studying the genome evolution after allopolyploidy. In this study, through screening BAC libraries and sequencing and annotating the targeted BAC clones, we generated orthologous genomic sequences surrounding the DEP1 locus, a major grain yield QTL in cultivated rice, from four Oryza polyploids of various ages and their likely diploid genome donors or close relatives. Based on sequenced DEP1 region and published data from three other genomic regions, we investigated the temporal evolutionary dynamics of four polyploid genomes at both genetic and expression levels. In the recently formed BBCC polyploid, Oryza minuta, genome dominance was not observed and its short-term responses to allopolyploidy are mainly manifested as a high proportion of homoeologous gene pairs showing unequal expression. This could partly be explained by parental legacy, rewiring of divergent regulatory networks and epigenetic modulation. Moreover, we detected an ongoing diploidization process in this genus, and suggest that the expression divergence driven by changes of selective constraint probably plays a big role in the long-term diploidization. These findings add novel insights into our understanding of genome evolution after allopolyploidy, and could facilitate crop improvements through hybridization and polyploidization.

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.

Cercis: A Non-polyploid Genomic Relic Within the Generally Polyploid Legume Family.

Based on evolutionary, phylogenomic, and synteny analyses of genome sequences for more than a dozen diverse legume species as well as analysis of chromosome counts across the legume family, we conclude that the genus Cercis provides a plausible model for an early evolutionary form of the legume genome. The small Cercis genus is in the earliest-diverging clade in the earliest-diverging legume subfamily (Cercidoideae). The Cercis genome is physically small, and has accumulated mutations at an unusually slow rate compared to other legumes. Chromosome counts across 477 legume genera, combined with phylogenetic reconstructions and histories of whole-genome duplications, suggest that the legume progenitor had 7 chromosomes - as does Cercis. We propose a model in which a legume progenitor, with 7 chromosomes, diversified into species that would become the Cercidoideae and the remaining legume subfamilies; then speciation in the Cercidoideae gave rise to the progenitor of the Cercis genus. There is evidence for a genome duplication in the remaining Cercidoideae, which is likely due to allotetraploidy involving hybridization between a Cercis progenitor and a second diploid species that existed at the time of the polyploidy event. Outside the Cercidoideae, a set of probably independent whole-genome duplications gave rise to the five other legume subfamilies, at least four of which have predominant counts of 12-14 chromosomes among their early-diverging taxa. An earlier study concluded that independent duplications occurred in the Caesalpinioideae, Detarioideae, and Papilionoideae. We conclude that Cercis may be unique among legumes in lacking evidence of polyploidy, a process that has shaped the genomes of all other legumes thus far investigated.

Segmental allopolyploidy in action: Increasing diversity through polyploid hybridization and homoeologous recombination.

PREMISE OF THE STUDY: The genetic bottleneck of polyploid formation can be mitigated by multiple origins, gene flow, and recombination among different lineages. In crop plants with limited origins, efforts to increase genetic diversity have limitations. Here we used lineage recombination to increase genetic diversity in peanut, an allotetraploid likely of single origin, by crossing with a novel allopolyploid genotype and selecting improved lines. METHODS: Single backcross progeny from cultivated peanut × wild species-derived allotetraploid cross were studied over successive generations. Using genetic assumptions that encompass segmental allotetraploidy, we used single nucleotide polymorphisms and whole-genome sequence data to infer genome structures. KEY RESULTS: Selected lines, despite a high proportion of wild alleles, are agronomically adapted, productive, and with improved disease resistances. Wild alleles mostly substituted homologous segments of the peanut genome. Regions of dispersed wild alleles, characteristic of gene conversion, also occurred. However, wild chromosome segments sometimes replaced cultivated peanut's homeologous subgenome; A. ipaënsis B sometimes replaced A. hypogaea A subgenome (~0.6%), and A. duranensis replaced A. hypogaea B subgenome segments (~2%). Furthermore, some subgenome regions historically lost in cultivated peanut were "recovered" by wild chromosome segments (effectively reversing the "polyploid ratchet"). These processes resulted in lines with new genome structure variations. CONCLUSIONS: Genetic diversity was introduced by wild allele introgression, and by introducing new genome structure variations. These results highlight the special possibilities of segmental allotetraploidy and of using lineage recombination to increase genetic diversity in peanut, likely mirroring what occurs in natural segmental allopolyploids with multiple origins.

Non-Additive Transcriptomic Responses to Inoculation with Rhizobia in a Young Allopolyploid Compared with Its Diploid Progenitors.

Root nodule symbioses (nodulation) and whole genome duplication (WGD, polyploidy) are both important phenomena in the legume family (Leguminosae). Recently, it has been proposed that polyploidy may have played a critical role in the origin or refinement of nodulation. However, while nodulation and polyploidy have been studied independently, there have been no direct studies of mechanisms affecting the interactions between these phenomena in symbiotic, nodule-forming species. Here, we examined the transcriptome-level responses to inoculation in the young allopolyploid Glycine dolichocarpa (T2) and its diploid progenitor species to identify underlying processes leading to the enhanced nodulation responses previously identified in T2. We assessed the differential expression of genes and, using weighted gene co-expression network analysis (WGCNA), identified modules associated with nodulation and compared their expression between species. These transcriptomic analyses revealed patterns of non-additive expression in T2, with evidence of transcriptional responses to inoculation that were distinct from one or both progenitors. These differential responses elucidate mechanisms underlying the nodulation-related differences observed between T2 and the diploid progenitors. Our results indicate that T2 has reduced stress-related transcription, coupled with enhanced transcription of modules and genes implicated in hormonal signaling, both of which are important for nodulation.

Karyotype Stability and Unbiased Fractionation in the Paleo-Allotetraploid Cucurbita Genomes.

The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Here, we report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetraploidization event in Cucurbita. We are able to partition the genome into two homoeologous subgenomes based on different genetic distances to melon, cucumber, and watermelon in the Benincaseae tribe. We estimate that the two diploid progenitors successively diverged from Benincaseae around 31 and 26 million years ago (Mya), respectively, and the allotetraploidization happened at some point between 26 Mya and 3 Mya, the estimated date when C. maxima and C. moschata diverged. The subgenomes have largely maintained the chromosome structures of their diploid progenitors. Such long-term karyotype stability after polyploidization has not been commonly observed in plant polyploids. The two subgenomes have retained similar numbers of genes, and neither subgenome is globally dominant in gene expression. Allele-specific expression analysis in the C. maxima × C. moschata interspecific F1 hybrid and their two parents indicates the predominance of trans-regulatory effects underlying expression divergence of the parents, and detects transgressive gene expression changes in the hybrid correlated with heterosis in important agronomic traits. Our study provides insights into polyploid genome evolution and valuable resources for genetic improvement of cucurbit crops.

Enhanced rhizobial symbiotic capacity in an allopolyploid species of Glycine (Leguminosae).

PREMISE OF THE STUDY: Previous studies have shown that polyploidy can alter biotic interactions, and it has been suggested that these effects may contribute to the increased ability for colonization of new habitats shown by many allopolyploids. Little is known, however, about the effects of allopolyploidy, which combines hybridity and genome doubling, on symbiotic interactions with rhizobial bacteria. METHODS: We examined interactions of the allopolyploid Glycine dolichocarpa (designated T2) with novel rhizobial partners, such as might occur in a context of colonization, and compared these with the responses of its diploid progenitors, G. tomentella (D3) and G. syndetika (D4). We assessed root hair response, nodule formation, nodule mass, nodule number, and plant biomass. KEY RESULTS: The allopolyploid (T2) showed a greater root hair deformation response when exposed to rhizobia, compared with either diploid. T2 had a greater probability of forming nodules with NGR234 compared with diploid D4, and greater total nodule mass per nodulated plant compared with diploid D3. T2 also had greater plant biomass responses to nitrogen and when exposed to NGR234. CONCLUSIONS: The allopolyploid is characterized by transgressive responses to rhizobia for some variables, while also combining certain parental diploid responses such that its capacity for interactions with rhizobia appears to be greater than for either diploid progenitor. This overall enhanced nodulation capacity and the ability to make greater gains from exposure to both rhizobia and additional nitrogen indicate a greater potential of the allopolyploid to benefit from these factors both generally and in a context of colonization.

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.

Multiple origins of BBCC allopolyploid species in the rice genus (Oryza).

In the rice genus (Oryza), about one half of the species are allopolyploids. These species are not only important resources for rice breeding but also provide a unique opportunity for studying evolution of polyploid species. In the present study, we sequenced four biparentally inherited nuclear loci and three maternally inherited chloroplast fragments from all diploid and tetraploid species with the B- and C-genome types in this genus. We detected at least three independent origins of three BC-genome tetraploid species. Specifically, the diploid O. punctata (B-genome) and O. officinalis (C-genome) were the parental progenitors of O. minuta and O. malampuzhaensis with O. punctata being the maternal donors, whereas the diploid O. punctata and O. eichingeri (C-genome) were the progenitors of tetraploid O. punctata with O. punctata being the paternal donor. Our relaxed clock analyses suggest that all the BBCC species originated within the last one million years, which is coincident with the severe climate oscillations occurred during the last ice age, implying the potential impact of climate change on their formations and dispersals. In addition, our results support previous taxonomic arguments that the tetraploid O. punctata might be better treated as a separate species (O. schweinfurthiana).

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.

Multiple polyploidy events in the early radiation of nodulating and nonnodulating legumes.

Unresolved questions about evolution of the large and diverse legume family include the timing of polyploidy (whole-genome duplication; WGDs) relative to the origin of the major lineages within the Fabaceae and to the origin of symbiotic nitrogen fixation. Previous work has established that a WGD affects most lineages in the Papilionoideae and occurred sometime after the divergence of the papilionoid and mimosoid clades, but the exact timing has been unknown. The history of WGD has also not been established for legume lineages outside the Papilionoideae. We investigated the presence and timing of WGDs in the legumes by querying thousands of phylogenetic trees constructed from transcriptome and genome data from 20 diverse legumes and 17 outgroup species. The timing of duplications in the gene trees indicates that the papilionoid WGD occurred in the common ancestor of all papilionoids. The earliest diverging lineages of the Papilionoideae include both nodulating taxa, such as the genistoids (e.g., lupin), dalbergioids (e.g., peanut), phaseoloids (e.g., beans), and galegoids (=Hologalegina, e.g., clovers), and clades with nonnodulating taxa including Xanthocercis and Cladrastis (evaluated in this study). We also found evidence for several independent WGDs near the base of other major legume lineages, including the Mimosoideae-Cassiinae-Caesalpinieae (MCC), Detarieae, and Cercideae clades. Nodulation is found in the MCC and papilionoid clades, both of which experienced ancestral WGDs. However, there are numerous nonnodulating lineages in both clades, making it unclear whether the phylogenetic distribution of nodulation is due to independent gains or a single origin followed by multiple losses.

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.

Climate niche modeling in the perennial Glycine (Leguminosae) allopolyploid complex.

PREMISE OF STUDY: Polyploid plants, when compared with diploids, show similar molecular, morphological, physiological, and ecological tendencies across unrelated groups, but the degree to which these form "rules" of polyploid evolution are unclear. The Glycine (Leguminosae) allopolyploid complex affords the opportunity to test whether polyploidy in similar genetic backgrounds produces similar effects on geographical range or climatic space. METHODS: We used information on locality presence of four closely related Glycine allopolyploid species and their diploid progenitors to build models of the potentially available Australian ranges based on climate using Maxent3.3.3k. Principal coordinate analysis was used to characterize the multidimensional climate space occupied by each species. KEY RESULTS: Each of the four Glycine allopolyploids showed intermediacy in potential geographical space and in ecological space, relative to its diploid progenitors. The four allopolyploids did not have consistently larger ranges than their progenitors, though all four occupied a portion of climate niche space not available to its progenitors. The polyploids also differed in their exploitation of potentially available geographical range. Australian ranges and environmental space did not correlate with greater colonizing ability in these polyploids. CONCLUSIONS: The four Glycine allopolyploids do not show many common range- or climate-related features, other than intermediacy. Thus, despite their similar genetic and evolutionary backgrounds, polyploidy has not produced convergent ecological effects.

Molecular phylogenetics of Amorpha (Fabaceae): an evaluation of monophyly, species relationships, and polyploid origins.

Amorpha L. (false indigos and lead plants) is a North American legume genus of 16 species of shrubs, which is most diverse in the southeastern United States and distinctive due to the reduction of the corolla to a single petal. Most species have limited distributions, but the tetraploid A. fruticosa species complex is widely distributed and its range overlaps those of all of the other species. Morphological variation in the genus is characterized by gradation of characters among species and it has been the subject of repeated taxonomic study due to the difficulty in delimiting species, especially among A. fruticosa and allies. This study presents the first phylogenetic and network analyses for evaluation of relationships amongst Amorpha species based on three non-coding plastome regions (trnD-trnT, trnH-psbA, petN-psbM) and two low-copy nuclear genes (CNGC5, minD). Plastid DNA analyses supported a monophyletic Amorpha with Parryella filifolia and Errazurizia rotundata as successive sister lineages; however, nuclear gene analyses supported the nesting of these two species and thus a paraphyletic Amorpha. Relationships among species of Amorpha were best resolved in the plastid DNA phylogeny and in most cases were concordant with expectations based on morphology. Relationships based on the nuclear gene phylogenies were less clear due to lack of informative variation (CNGC5) or conflict in the data set (minD). The origins of A. fruticosa were unclear, but the plastid phylogeny revealed that this species shares the same or similar plastid haplotype as other species in a geographic region. Putative recombination of diploid species' alleles was evident in the minD-like network. Phenotypic plasticity in combination with gene flow into this species from different diploids, or even tetraploids, across its range may account for the incredible morphological diversity of the A. fruticosa species complex. Putative progenitors for two other suspected allotetraploid species, A. confusa and A. crenulata, were identified as A. fruticosa and A. herbacea.