The RNA binding protein EHD6 recruits the m6A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering.

N6-methyladenosine (m6A) is one of the most abundant modifications in eukaryotic mRNA, but the comprehensive biological functionality continues to be a subject for exploration. In this study, we identified and characterized a new flowering-promoting gene EARLY HEADING DATE6 (EHD6) in rice. EHD6 encodes an RNA recognition motif (RRM)-containing RNA binding protein that is localized in the non-membranous cytoplasm ribonucleoprotein (RNP) granules and can bind both m6A-modified RNA and unmodified RNA indiscriminately. We found that EHD6 can physically interact with YTH07, a YTH (YT521-B homology) domain containing m6A reader, and their interaction enhances the binding of m6A-modified RNA and triggers relocation of a part of YTH07 from the cytoplasm into RNP granules through phase-separated condensation. Within these condensates, the mRNA of a rice flowering repressor, CONSTANS-like 4 (OsCOL4), becomes sequestered, leading to a reduction in its protein abundance and thus affect flowering through the Early heading date 1 pathway. Our results not only shed new light on the molecular mechanism of efficient m6A recognition by the collaboration between the RNA binding protein and YTH family m6A reader, but also uncovers a potential m6A mediated translation regulation through phase-separated ribonucleoprotein condensation in rice.

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.

An Integrated Fast Hough Transform for Multidimensional Data.

Line, plane and hyperplane detection in multidimensional data has many applications in computer vision and artificial intelligence. We propose Integrated Fast Hough Transform (IFHT), a highly-efficient multidimensional Hough transform algorithm based on a new mathematical model. The parameter space of IFHT can be represented with a single k-tree to support hierarchical storage and "coarse-to-fine" search strategy. IFHT essentially changes the least square data-fitting in Li's Fast Hough transform (FHT) to the total least squares data-fitting, in which observational errors across all dimensions are taken into account, thus more practical and more resistant to data noise. It has practically resolved the problem of decreased precision of FHT for target objects mapped to boundaries between accumulators in the parameter space. In addition, it enables a straightforward visualization of the parameter space which not only provides intuitive insight on the number of objects in the data, but also helps with tuning the parameters and combining multiple instances if needed. In all simulated data with different levels of noise and parameters, IFHT surpasses Li's Fast Hough transform in terms of robustness and precision significantly.

GALA: a computational framework for de novo chromosome-by-chromosome assembly with long reads.

High-quality genome assembly has wide applications in genetics and medical studies. However, it is still very challenging to achieve gap-free chromosome-scale assemblies using current workflows for long-read platforms. Here we report on GALA (Gap-free long-read Assembly tool), a computational framework for chromosome-based sequencing data separation and de novo assembly implemented through a multi-layer graph that identifies discordances within preliminary assemblies and partitions the data into chromosome-scale scaffolding groups. The subsequent independent assembly of each scaffolding group generates a gap-free assembly likely free from the mis-assembly errors which usually hamper existing workflows. This flexible framework also allows us to integrate data from various technologies, such as Hi-C, genetic maps, and even motif analyses to generate gap-free chromosome-scale assemblies. As a proof of principle we de novo assemble the C. elegans genome using combined PacBio and Nanopore sequencing data and a rice cultivar genome using Nanopore sequencing data from publicly available datasets. We also demonstrate the proposed method's applicability with a gap-free assembly of the human genome using PacBio high-fidelity (HiFi) long reads. Thus, our method enables straightforward assembly of genomes with multiple data sources and overcomes barriers that at present restrict the application of de novo genome assembly technology.

Gene expression evolution in pattern-triggered immunity within Arabidopsis thaliana and across Brassicaceae species.

Plants recognize surrounding microbes by sensing microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). Despite their significance for microbial control, the evolution of PTI responses remains largely uncharacterized. Here, by employing comparative transcriptomics of six Arabidopsis thaliana accessions and three additional Brassicaceae species to investigate PTI responses, we identified a set of genes that commonly respond to the MAMP flg22 and genes that exhibit species-specific expression signatures. Variation in flg22-triggered transcriptome responses across Brassicaceae species was incongruent with their phylogeny, while expression changes were strongly conserved within A. thaliana. We found the enrichment of WRKY transcription factor binding sites in the 5'-regulatory regions of conserved and species-specific responsive genes, linking the emergence of WRKY-binding sites with the evolution of gene expression patterns during PTI. Our findings advance our understanding of the evolution of the transcriptome during biotic stress.

Beyond flowering time: diverse roles of an APETALA2-like transcription factor in shoot architecture and perennial traits.

Polycarpic perennials maintain vegetative growth after flowering. PERPETUAL FLOWERING 1 (PEP1), the orthologue of FLOWERING LOCUS C (FLC) in Arabis alpina regulates flowering and contributes to polycarpy in a vernalisation-dependent pathway. pep1 mutants do not require vernalisation to flower and have reduced return to vegetative growth as all of their axillary branches become reproductive. To identify additional genes that regulate flowering and contribute to perennial traits we performed an enhancer screen of pep1. Using mapping-by-sequencing, we cloned a mutant (enhancer of pep1-055, eop055), performed transcriptome analysis and physiologically characterised the role it plays on perennial traits in an introgression line carrying the eop055 mutation and a functional PEP1 wild-type allele. eop055 flowers earlier than pep1 and carries a lesion in the A. alpina orthologue of the APETALA2 (AP2)-like gene, TARGET OF EAT2 (AaTOE2). AaTOE2 is a floral repressor and acts upstream of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 5 (AaSPL5). In the wild-type background, which requires cold treatment to flower, AaTOE2 regulates the age-dependent response to vernalisation. In addition, AaTOE2 ensures the maintenance of vegetative growth by delaying axillary meristem initiation and repressing flowering of axillary buds before and during cold exposure. We conclude that AaTOE2 is instrumental in fine tuning different developmental traits in the perennial life cycle of A. alpina.

Fine-scale empirical data on niche divergence and homeolog expression patterns in an allopolyploid and its diploid progenitor species.

Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine-scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors. We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing. We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally. These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

A WOX/Auxin Biosynthesis Module Controls Growth to Shape Leaf Form.

A key challenge in biology is to understand how the regional control of cell growth gives rise to final organ forms. Plant leaves must coordinate growth along both the proximodistal and mediolateral axes to produce their final shape. However, the cell-level mechanisms controlling this coordination remain largely unclear. Here, we show that, in A. thaliana, WOX5, one of the WUSCHEL-RELATED HOMEOBOX (WOX) family of homeobox genes, acts redundantly with WOX1 and WOX3 (PRESSED FLOWER [PRS]) to control leaf shape. Through genetics and hormone measurements, we find that these WOXs act in part through the regional control of YUCCA (YUC) auxin biosynthetic gene expression along the leaf margin. The requirement for WOX-mediated YUC expression in patterning of leaf shape cannot be bypassed by the epidermal expression of YUC, indicating that the precise domain of auxin biosynthesis is important for leaf form. Using time-lapse growth analysis, we demonstrate that WOX-mediated auxin biosynthesis organizes a proximodistal growth gradient that promotes lateral growth and consequently the characteristic ellipsoid A. thaliana leaf shape. We also provide evidence that WOX proteins shape the proximodistal gradient of differentiation by inhibiting differentiation proximally in the leaf blade and promoting it distally. This regulation allows sustained growth of the blade and enables a leaf to attain its final form. In conclusion, we show that the WOX/auxin regulatory module shapes leaf form by coordinating growth along the proximodistal and mediolateral leaf axes.

Complement Genome Annotation Lift Over Using a Weighted Sequence Alignment Strategy.

With the broad application of high-throughput sequencing, more whole-genome resequencing data and de novo assemblies of natural populations are becoming available. For a particular species, in general, only the reference genome is well established and annotated. Computational tools based on sequence alignment have been developed to investigate the gene models of individuals belonging to the same or closely related species. During this process, inconsistent alignment often obscures genome annotation lift over and leads to improper functional impact prediction for a genomic variant, especially in plant species. Here, we proposed the zebraic striped dynamic programming algorithm, which provides different weights to genetic features to refine genome annotation lift over. Testing of our zebraic striped dynamic programming algorithm on both plant and animal genomic data showed complementation to standard sequence approach for highly diverse individuals. Using the lift over genome annotation as anchors, a base-pair resolution genome-wide sequence alignment and variant calling pipeline for de novo assembly has been implemented in the GEAN software. GEAN could be used to compare haplotype diversity, refine the genetic variant functional annotation, annotate de novo assembly genome sequence, detect homologous syntenic blocks, improve the quantification of gene expression levels using RNA-seq data, and unify genomic variants for population genetic analysis. We expect that GEAN will be a standard tool for the coming of age of de novo assembly population genetics.

Autoregulation of RCO by Low-Affinity Binding Modulates Cytokinin Action and Shapes Leaf Diversity.

Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A. thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C. hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species.

Photoreceptor Activity Contributes to Contrasting Responses to Shade in Cardamine and Arabidopsis Seedlings.

Plants have evolved two major ways to deal with nearby vegetation or shade: avoidance and tolerance. Moreover, some plants respond to shade in different ways; for example, Arabidopsis (Arabidopsis thaliana) undergoes an avoidance response to shade produced by vegetation, but its close relative Cardamine hirsuta tolerates shade. How plants adopt opposite strategies to respond to the same environmental challenge is unknown. Here, using a genetic strategy, we identified the C. hirsuta slender in shade1 mutants, which produce strongly elongated hypocotyls in response to shade. These mutants lack the phytochrome A (phyA) photoreceptor. Our findings suggest that C. hirsuta has evolved a highly efficient phyA-dependent pathway that suppresses hypocotyl elongation when challenged by shade from nearby vegetation. This suppression relies, at least in part, on stronger phyA activity in C. hirsuta; this is achieved by increased ChPHYA expression and protein accumulation combined with a stronger specific intrinsic repressor activity. We suggest that modulation of photoreceptor activity is a powerful mechanism in nature to achieve physiological variation (shade tolerance versus avoidance) for species to colonize different habitats.

Recently duplicated sesterterpene (C25) gene clusters in Arabidopsis thaliana modulate root microbiota.

Land plants co-speciate with a diversity of continually expanding plant specialized metabolites (PSMs) and root microbial communities (microbiota). Homeostatic interactions between plants and root microbiota are essential for plant survival in natural environments. A growing appreciation of microbiota for plant health is fuelling rapid advances in genetic mechanisms of controlling microbiota by host plants. PSMs have long been proposed to mediate plant and single microbe interactions. However, the effects of PSMs, especially those evolutionarily new PSMs, on root microbiota at community level remain to be elucidated. Here, we discovered sesterterpenes in Arabidopsis thaliana, produced by recently duplicated prenyltransferase-terpene synthase (PT-TPS) gene clusters, with neo-functionalization. A single-residue substitution played a critical role in the acquisition of sesterterpene synthase (sesterTPS) activity in Brassicaceae plants. Moreover, we found that the absence of two root-specific sesterterpenoids, with similar chemical structure, significantly affected root microbiota assembly in similar patterns. Our results not only demonstrate the sensitivity of plant microbiota to PSMs but also establish a complete framework of host plants to control root microbiota composition through evolutionarily dynamic PSMs.

Resolving the backbone of the Brassicaceae phylogeny for investigating trait diversity.

The Brassicaceae family comprises c. 4000 species including economically important crops and the model plant Arabidopsis thaliana. Despite their importance, the relationships among major lineages in the family remain unresolved, hampering comparative research. Here, we inferred a Brassicaceae phylogeny using newly generated targeted enrichment sequence data of 1827 exons (> 940 000 bases) representing 63 species, as well as sequenced genome data of 16 species, together representing 50 of the 52 currently recognized Brassicaceae tribes. A third of the samples were derived from herbarium material, facilitating broad taxonomic coverage of the family. Six major clades formed successive sister groups to the rest of Brassicaceae. We also recovered strong support for novel relationships among tribes, and resolved the position of 16 taxa previously not assigned to a tribe. The broad utility of these phylogenetic results is illustrated through a comparative investigation of genome-wide expression signatures that distinguish simple from complex leaves in Brassicaceae. Our study provides an easily extendable dataset for further advances in Brassicaceae systematics and a timely higher-level phylogenetic framework for a wide range of comparative studies of multiple traits in an intensively investigated group of plants.

LMI1 homeodomain protein regulates organ proportions by spatial modulation of endoreduplication.

How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.

Recovery of novel association loci in Arabidopsis thaliana and Drosophila melanogaster through leveraging INDELs association and integrated burden test.

Short insertions, deletions (INDELs) and larger structural variants have been increasingly employed in genetic association studies, but few improvements over SNP-based association have been reported. In order to understand why this might be the case, we analysed two publicly available datasets and observed that 63% of INDELs called in A. thaliana and 64% in D. melanogaster populations are misrepresented as multiple alleles with different functional annotations, i.e. where the same underlying variant is represented by inconsistent alignments leading to different variant calls. To address this issue, we have developed the software Irisas to reclassify and re-annotate these variants, which we then used for single-locus tests of association. We also integrated them to predict the functional impact of SNPs, INDELs, and structural variants for burden testing. Using both approaches, we re-analysed the genetic architecture of complex traits in A. thaliana and D. melanogaster. Heritability analysis using SNPs alone explained on average 27% and 19% of phenotypic variance for A. thaliana and D. melanogaster respectively. Our method explained an additional 11% and 3%, respectively. We also identified novel trait loci that previous SNP-based association studies failed to map, and which contain established candidate genes. Our study shows the value of the association test with INDELs and integrating multiple types of variants in association studies in plants and animals.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii, isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions.

The Cardamine hirsuta genome offers insight into the evolution of morphological diversity.

Finding causal relationships between genotypic and phenotypic variation is a key focus of evolutionary biology, human genetics and plant breeding. To identify genome-wide patterns underlying trait diversity, we assembled a high-quality reference genome of Cardamine hirsuta, a close relative of the model plant Arabidopsis thaliana. We combined comparative genome and transcriptome analyses with the experimental tools available in C. hirsuta to investigate gene function and phenotypic diversification. Our findings highlight the prevalent role of transcription factors and tandem gene duplications in morphological evolution. We identified a specific role for the transcriptional regulators PLETHORA5/7 in shaping leaf diversity and link tandem gene duplication with differential gene expression in the explosive seed pod of C. hirsuta. Our work highlights the value of comparative approaches in genetically tractable species to understand the genetic basis for evolutionary change.