Abstract: Legumes form symbiosis with rhizobium leading to the development of nitrogen-fixing nodules. By integrating single-nucleus and spatial transcriptomics, we established a cell atlas of soybean nodules and roots. In central infected zones of nodules, we found that uninfected cells specialize into functionally distinct subgroups during nodule development, and revealed a transitional subtype of infected cells with enriched nodulation-related genes. Overall, our results provide a single-cell perspective for understanding rhizobium-legume symbiosis.

Abstract: Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

Abstract: Abstract Peatlands are crucial sinks for atmospheric carbon but are critically threatened due to warming climates. Sphagnum (peat moss) species are keystone members of peatland communities where they actively engineer hyperacidic conditions, which improves their competitive advantage and accelerates ecosystem-level carbon sequestration. To dissect the molecular and physiological sources of this unique biology, we generated chromosome-scale genomes of two Sphagnum species: S. divinum and S. angustifolium . Sphagnum genomes show no gene colinearity with any other reference genome to date, demonstrating that Sphagnum represents an unsampled lineage of land plant evolution. The genomes also revealed an average recombination rate an order of magnitude higher than vascular land plants and short putative U/V sex chromosomes. These newly described sex chromosomes interact with autosomal loci that significantly impact growth across diverse pH conditions. This discovery demonstrates that the ability of Sphagnum to sequester carbon in acidic peat bogs is mediated by interactions between sex, autosomes and environment.

Abstract: Retrieving the complex responses of individual cells in the native three-dimensional tissue context is crucial for a complete understanding of tissue functions. Here, we present PHYTOMap (plant hybridization-based targeted observation of gene expression map), a multiplexed fluorescence in situ hybridization method that enables single-cell and spatial analysis of gene expression in whole-mount plant tissue in a transgene-free manner and at low cost. We applied PHYTOMap to simultaneously analyse 28 cell-type marker genes in Arabidopsis roots and successfully identified major cell types, demonstrating that our method can substantially accelerate the spatial mapping of marker genes defined in single-cell RNA-sequencing datasets in complex plant tissue.

Abstract: Abstract Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Abstract: Abstract Stem vascular cambium cells in forest trees produce wood for materials and energy. WOX4 affects the proliferation of such cells in Populus . Here we show that Ptr WOX4a is the most highly expressed stem vascular-cambium-specific (VCS) gene in P. trichocarpa , and its expression is controlled by the product of the second most highly expressed VCS gene, Ptr VCS2 , encoding a zinc finger protein. PtrVCS2 binds to the Ptr WOX4a promoter as part of a PtrWOX13a–PtrVCS2–PtrGCN5-1–PtrADA2b-3 protein tetramer. PtrVCS2 prevented the interaction between PtrGCN5-1 and PtrADA2b-3, resulting in H3K9, H3K14 and H3K27 hypoacetylation at the Ptr WOX4a promoter, which led to fewer cambium cell layers. These effects on cambium cell proliferation were consistent across more than 20 sets of transgenic lines overexpressing individual genes, gene-edited mutants and RNA interference lines in P. trichocarpa . We propose that the tetramer–Ptr WOX4a system may coordinate genetic and epigenetic regulation to maintain normal vascular cambium development for wood formation.

Abstract: Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts. The study describes the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in plants by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components.

Abstract: Stress can have long-lasting impacts on plants. Here we report the long-term effects of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis. Three weeks after transient JA signalling, 5-week-old plants retained induced resistance (IR) against herbivory but showed increased susceptibility to pathogens. Transcriptome analysis revealed long-term priming and/or upregulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR was associated with shifts in glucosinolate composition and required MYC2/3/4 transcription factors, RNA-directed DNA methylation, the DNA demethylase ROS1 and the small RNA (sRNA)-binding protein AGO1. Although methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled genes, JA-treated plants were specifically enriched with hypomethylated ATREP2 transposable elements (TEs). Epigenomic characterization of mutants and transgenic lines revealed that ATREP2 TEs are regulated by RdDM and ROS1 and produce 21 nt sRNAs that bind to nuclear AGO1. Since ATREP2 TEs are enriched with sequences from IR-related defence genes, our results suggest that AGO1-associated sRNAs from hypomethylated ATREP2 TEs trans-regulate long-lasting memory of JA-dependent immunity. Jasmonic acid (JA) is involved in defence against herbivores. Once the plant is attacked, the memory of aggression can linger for weeks, including in newly formed leaves. The authors investigate what the induced changes in the transcriptome and methylome are, and uncouple short- and long-term effects of JA.

Abstract: Plant terrestrialization brought forth the land plants (embryophytes). Embryophytes account for most of the biomass on land and evolved from streptophyte algae in a singular event. Recent advances have unravelled the first full genomes of the closest algal relatives of land plants; among the first such species was Mesotaenium endlicherianum. Here we used fine-combed RNA sequencing in tandem with a photophysiological assessment on Mesotaenium exposed to a continuous range of temperature and light cues. Our data establish a grid of 42 different conditions, resulting in 128 transcriptomes and ~1.5 Tbp (~9.9 billion reads) of data to study the combinatory effects of stress response using clustering along gradients. Mesotaenium shares with land plants major hubs in genetic networks underpinning stress response and acclimation. Our data suggest that lipid droplet formation and plastid and cell wall-derived signals have denominated molecular programmes since more than 600 million years of streptophyte evolution-before plants made their first steps on land.

Abstract: Symbiotic interactions such as the nitrogen-fixing root nodule symbiosis (RNS) have structured ecosystems during the evolution of life. Here we aimed at reconstructing ancestral and intermediate steps that shaped RNS observed in extant flowering plants. We compared the symbiotic transcriptomic responses of nine host plants, including the mimosoid legume Mimosa pudica for which we assembled a chromosome-level genome. We reconstructed the ancestral RNS transcriptome composed of most known symbiotic genes together with hundreds of novel candidates. Cross-referencing with transcriptomic data in response to experimentally evolved bacterial strains with gradual symbiotic proficiencies, we found the response to bacterial signals, nodule infection, nodule organogenesis and nitrogen fixation to be ancestral. By contrast, the release of symbiosomes was associated with recently evolved genes encoding small proteins in each lineage. We demonstrate that the symbiotic response was mostly in place in the most recent common ancestor of the RNS-forming species more than 90 million years ago.

Abstract: Although climate change projections indicate significant threats to terrestrial biodiversity, the effects are much more profound and striking in the marine environment. Here we explore how different facets of locally distinctive α- and β-diversity (changes in spatial composition) of seagrasses will respond to future climate change scenarios across the globe and compare their coverage with the existing network of marine protected areas. By using species distribution modelling and a dated phylogeny, we predict widespread reductions in species' range sizes that will result in increases in seagrass weighted and phylogenetic endemism. These projected increases of endemism will result in divergent shifts in the spatial composition of β-diversity leading to differentiation in some areas and the homogenization of seagrass communities in other regions. Regardless of the climate scenario, the potential hotspots of these projected shifts in seagrass α- and β-diversity are predicted to occur outside the current network of marine protected areas, providing new priority areas for future conservation planning that incorporate seagrasses. Our findings report responses of species to future climate for a group that is currently under represented in climate change assessments yet crucial in maintaining marine food chains and providing habitat for a wide range of marine biodiversity.

Abstract: Abstract Here an improved carrot reference genome and resequencing of 630 carrot accessions were used to investigate carrot domestication and improvement. The study demonstrated that carrot was domesticated during the Early Middle Ages in the region spanning western Asia to central Asia, and orange carrot was selected during the Renaissance period, probably in western Europe. A progressive reduction of genetic diversity accompanied this process. Genes controlling circadian clock/flowering and carotenoid accumulation were under selection during domestication and improvement. Three recessive genes, at the REC , Or and Y2 quantitative trait loci, were essential to select for the high α- and β-carotene orange phenotype. All three genes control high α- and β-carotene accumulation through molecular mechanisms that regulate the interactions between the carotenoid biosynthetic pathway, the photosynthetic system and chloroplast biogenesis. Overall, this study elucidated carrot domestication and breeding history and carotenoid genetics at a molecular level.