Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.

Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.

Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.

Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.

Predicting yield of individual field-grown rapeseed plants from rosette-stage leaf gene expression.

In the plant sciences, results of laboratory studies often do not translate well to the field. To help close this lab-field gap, we developed a strategy for studying the wiring of plant traits directly in the field, based on molecular profiling and phenotyping of individual plants. Here, we use this single-plant omics strategy on winter-type Brassica napus (rapeseed). We investigate to what extent early and late phenotypes of field-grown rapeseed plants can be predicted from their autumnal leaf gene expression, and find that autumnal leaf gene expression not only has substantial predictive power for autumnal leaf phenotypes but also for final yield phenotypes in spring. Many of the top predictor genes are linked to developmental processes known to occur in autumn in winter-type B. napus accessions, such as the juvenile-to-adult and vegetative-to-reproductive phase transitions, indicating that the yield potential of winter-type B. napus is influenced by autumnal development. Our results show that single-plant omics can be used to identify genes and processes influencing crop yield in the field.

ksrates: positioning whole-genome duplications relative to speciation events in KS distributions.

SUMMARY: We present ksrates, a user-friendly command-line tool to position ancient whole-genome duplication events with respect to speciation events in a phylogeny by comparing paralog and ortholog KS distributions derived from genomic or transcriptomic sequences, while adjusting for substitution rate differences among the lineages involved. AVAILABILITY AND IMPLEMENTATION: ksrates is implemented in Python 3 and as a Nextflow pipeline. The source code, Singularity and Docker containers, documentation and tutorial are available via https://github.com/VIB-PSB/ksrates. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Drought affects the rate and duration of organ growth but not inter-organ growth coordination.

Drought at flowering and grain filling greatly reduces maize (Zea mays) yield. Climate change is causing earlier and longer-lasting periods of drought, which affect the growth of multiple maize organs throughout development. To study how long periods of water deficit impact the dynamic nature of growth, and to determine how these relate to reproductive drought, we employed a high-throughput phenotyping platform featuring precise irrigation, imaging systems, and image-based biomass estimations. Prolonged drought resulted in a reduction of growth rate of individual organs-though an extension of growth duration partially compensated for this-culminating in lower biomass and delayed flowering. However, long periods of drought did not affect the highly organized succession of maximal growth rates of the distinct organs, i.e. leaves, stems, and ears. Two drought treatments negatively affected distinct seed yield components: Prolonged drought mainly reduced the number of spikelets, and drought during the reproductive period increased the anthesis-silking interval. The identification of these divergent biomass and yield components, which were affected by the shift in duration and intensity of drought, will facilitate trait-specific breeding toward future climate-resilient crops.

Using single-plant-omics in the field to link maize genes to functions and phenotypes.

Most of our current knowledge on plant molecular biology is based on experiments in controlled laboratory environments. However, translating this knowledge from the laboratory to the field is often not straightforward, in part because field growth conditions are very different from laboratory conditions. Here, we test a new experimental design to unravel the molecular wiring of plants and study gene-phenotype relationships directly in the field. We molecularly profiled a set of individual maize plants of the same inbred background grown in the same field and used the resulting data to predict the phenotypes of individual plants and the function of maize genes. We show that the field transcriptomes of individual plants contain as much information on maize gene function as traditional laboratory-generated transcriptomes of pooled plant samples subject to controlled perturbations. Moreover, we show that field-generated transcriptome and metabolome data can be used to quantitatively predict individual plant phenotypes. Our results show that profiling individual plants in the field is a promising experimental design that could help narrow the lab-field gap.

A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Reciprocally Retained Genes in the Angiosperm Lineage Show the Hallmarks of Dosage Balance Sensitivity.

In several organisms, particular functional categories of genes, such as regulatory and complex-forming genes, are preferentially retained after whole-genome multiplications but rarely duplicate through small-scale duplication, a pattern referred to as reciprocal retention. This peculiar duplication behavior is hypothesized to stem from constraints on the dosage balance between the genes concerned and their interaction context. However, the evidence for a relationship between reciprocal retention and dosage balance sensitivity remains fragmentary. Here, we identified which gene families are most strongly reciprocally retained in the angiosperm lineage and studied their functional and evolutionary characteristics. Reciprocally retained gene families exhibit stronger sequence divergence constraints and lower rates of functional and expression divergence than other gene families, suggesting that dosage balance sensitivity is a general characteristic of reciprocally retained genes. Gene families functioning in regulatory and signaling processes are much more strongly represented at the top of the reciprocal retention ranking than those functioning in multiprotein complexes, suggesting that regulatory imbalances may lead to stronger fitness effects than classical stoichiometric protein complex imbalances. Finally, reciprocally retained duplicates are often subject to dosage balance constraints for prolonged evolutionary times, which may have repercussions for the ease with which genome multiplications can engender evolutionary innovation.

The Origin of Floral Organ Identity Quartets.

The origin of flowers has puzzled plant biologists ever since Darwin referred to their sudden appearance in the fossil record as an abominable mystery. Flowers are considered to be an assembly of protective, attractive, and reproductive male and female leaf-like organs. Their origin cannot be understood by a morphological comparison to gymnosperms, their closest relatives, which develop separate male or female cones. Despite these morphological differences, gymnosperms and angiosperms possess a similar genetic toolbox consisting of phylogenetically related MADS domain proteins. Using ancestral MADS domain protein reconstruction, we trace the evolution of organ identity quartets along the stem lineage of crown angiosperms. We provide evidence that current floral quartets specifying male organ identity, which consist of four types of subunits, evolved from ancestral complexes of two types of subunits through gene duplication and integration of SEPALLATA proteins just before the origin of flowering plants. Our results suggest that protein interaction changes underlying this compositional shift were the result of a gradual and reversible evolutionary trajectory. Modeling shows that such compositional changes may have facilitated the evolution of the perfect, bisexual flower.

Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms.

Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of "gene duplicability" is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes.

Leaf Growth Response to Mild Drought: Natural Variation in Arabidopsis Sheds Light on Trait Architecture.

Plant growth and crop yield are negatively affected by a reduction in water availability. However, a clear understanding of how growth is regulated under nonlethal drought conditions is lacking. Recent advances in genomics, phenomics, and transcriptomics allow in-depth analysis of natural variation. In this study, we conducted a detailed screening of leaf growth responses to mild drought in a worldwide collection of Arabidopsis thaliana accessions. The genetic architecture of the growth responses upon mild drought was investigated by subjecting the different leaf growth phenotypes to genome-wide association mapping and by characterizing the transcriptome of young developing leaves. Although no major effect locus was found to be associated with growth in mild drought, the transcriptome analysis delivered further insight into the natural variation of transcriptional responses to mild drought in a specific tissue. Coexpression analysis indicated the presence of gene clusters that co-vary over different genetic backgrounds, among others a cluster of genes with important regulatory functions in the growth response to osmotic stress. It was found that the occurrence of a mild drought stress response in leaves can be inferred with high accuracy across accessions based on the expression profile of 283 genes. A genome-wide association study on the expression data revealed that trans regulation seems to be more important than cis regulation in the transcriptional response to environmental perturbations.

The reduction in maize leaf growth under mild drought affects the transition between cell division and cell expansion and cannot be restored by elevated gibberellic acid levels.

Growth is characterized by the interplay between cell division and cell expansion, two processes that occur separated along the growth zone at the maize leaf. To gain further insight into the transition between cell division and cell expansion, conditions were investigated in which the position of this transition zone was positively or negatively affected. High levels of gibberellic acid (GA) in plants overexpressing the GA biosynthesis gene GA20-OXIDASE (GA20OX-1OE ) shifted the transition zone more distally, whereas mild drought, which is associated with lowered GA biosynthesis, resulted in a more basal positioning. However, the increased levels of GA in the GA20OX-1OE line were insufficient to convey tolerance to the mild drought treatment, indicating that another mechanism in addition to lowered GA levels is restricting growth during drought. Transcriptome analysis with high spatial resolution indicated that mild drought specifically induces a reprogramming of transcriptional regulation in the division zone. 'Leaf Growth Viewer' was developed as an online searchable tool containing the high-resolution data.

Analysis of 41 plant genomes supports a wave of successful genome duplications in association with the Cretaceous-Paleogene boundary.

Ancient whole-genome duplications (WGDs), also referred to as paleopolyploidizations, have been reported in most evolutionary lineages. Their attributed role remains a major topic of discussion, ranging from an evolutionary dead end to a road toward evolutionary success, with evidence supporting both fates. Previously, based on dating WGDs in a limited number of plant species, we found a clustering of angiosperm paleopolyploidizations around the Cretaceous-Paleogene (K-Pg) extinction event about 66 million years ago. Here we revisit this finding, which has proven controversial, by combining genome sequence information for many more plant lineages and using more sophisticated analyses. We include 38 full genome sequences and three transcriptome assemblies in a Bayesian evolutionary analysis framework that incorporates uncorrelated relaxed clock methods and fossil uncertainty. In accordance with earlier findings, we demonstrate a strongly nonrandom pattern of genome duplications over time with many WGDs clustering around the K-Pg boundary. We interpret these results in the context of recent studies on invasive polyploid plant species, and suggest that polyploid establishment is promoted during times of environmental stress. We argue that considering the evolutionary potential of polyploids in light of the environmental and ecological conditions present around the time of polyploidization could mitigate the stark contrast in the proposed evolutionary fates of polyploids.

Combined Large-Scale Phenotyping and Transcriptomics in Maize Reveals a Robust Growth Regulatory Network.

Leaves are vital organs for biomass and seed production because of their role in the generation of metabolic energy and organic compounds. A better understanding of the molecular networks underlying leaf development is crucial to sustain global requirements for food and renewable energy. Here, we combined transcriptome profiling of proliferative leaf tissue with in-depth phenotyping of the fourth leaf at later stages of development in 197 recombinant inbred lines of two different maize (Zea mays) populations. Previously, correlation analysis in a classical biparental mapping population identified 1,740 genes correlated with at least one of 14 traits. Here, we extended these results with data from a multiparent advanced generation intercross population. As expected, the phenotypic variability was found to be larger in the latter population than in the biparental population, although general conclusions on the correlations among the traits are comparable. Data integration from the two diverse populations allowed us to identify a set of 226 genes that are robustly associated with diverse leaf traits. This set of genes is enriched for transcriptional regulators and genes involved in protein synthesis and cell wall metabolism. In order to investigate the molecular network context of the candidate gene set, we integrated our data with publicly available functional genomics data and identified a growth regulatory network of 185 genes. Our results illustrate the power of combining in-depth phenotyping with transcriptomics in mapping populations to dissect the genetic control of complex traits and present a set of candidate genes for use in biomass improvement.

A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants.

A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta.

Predicting gene function from uncontrolled expression variation among individual wild-type Arabidopsis plants.

Gene expression profiling studies are usually performed on pooled samples grown under tightly controlled experimental conditions to suppress variability among individuals and increase experimental reproducibility. In addition, to mask unwanted residual effects, the samples are often subjected to relatively harsh treatments that are unrealistic in a natural context. Here, we show that expression variations among individual wild-type Arabidopsis thaliana plants grown under the same macroscopic growth conditions contain as much information on the underlying gene network structure as expression profiles of pooled plant samples under controlled experimental perturbations. We advocate the use of subtle uncontrolled variations in gene expression between individuals to uncover functional links between genes and unravel regulatory influences. As a case study, we use this approach to identify ILL6 as a new regulatory component of the jasmonate response pathway.

Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants.

The importance of gene gain through duplication has long been appreciated. In contrast, the importance of gene loss has only recently attracted attention. Indeed, studies in organisms ranging from plants to worms and humans suggest that duplication of some genes might be better tolerated than that of others. Here we have undertaken a large-scale study to investigate the existence of duplication-resistant genes in the sequenced genomes of 20 flowering plants. We demonstrate that there is a large set of genes that is convergently restored to single-copy status following multiple genome-wide and smaller scale duplication events. We rule out the possibility that such a pattern could be explained by random gene loss only and therefore propose that there is selection pressure to preserve such genes as singletons. This is further substantiated by the observation that angiosperm single-copy genes do not comprise a random fraction of the genome, but instead are often involved in essential housekeeping functions that are highly conserved across all eukaryotes. Furthermore, single-copy genes are generally expressed more highly and in more tissues than non-single-copy genes, and they exhibit higher sequence conservation. Finally, we propose different hypotheses to explain their resistance against duplication.

Reconstruction of ancestral metabolic enzymes reveals molecular mechanisms underlying evolutionary innovation through gene duplication.

Gene duplications are believed to facilitate evolutionary innovation. However, the mechanisms shaping the fate of duplicated genes remain heavily debated because the molecular processes and evolutionary forces involved are difficult to reconstruct. Here, we study a large family of fungal glucosidase genes that underwent several duplication events. We reconstruct all key ancestral enzymes and show that the very first preduplication enzyme was primarily active on maltose-like substrates, with trace activity for isomaltose-like sugars. Structural analysis and activity measurements on resurrected and present-day enzymes suggest that both activities cannot be fully optimized in a single enzyme. However, gene duplications repeatedly spawned daughter genes in which mutations optimized either isomaltase or maltase activity. Interestingly, similar shifts in enzyme activity were reached multiple times via different evolutionary routes. Together, our results provide a detailed picture of the molecular mechanisms that drove divergence of these duplicated enzymes and show that whereas the classic models of dosage, sub-, and neofunctionalization are helpful to conceptualize the implications of gene duplication, the three mechanisms co-occur and intertwine.