Laticifers are specialized secretory cells that synthesize and store latex and provide a model for studying cellular specialization in plant metabolism. Euphorbia lathyris L. possesses non-articulated laticifers that produce triterpenoid-rich latex, but the mechanistic basis of laticifer specialization and latex metabolism remains unclear. Here, we generate the single-nucleus transcriptomic landscape of E. lathyris leaves and resolve a continuous laticifer trajectory with progressive activation of the mevalonate-derived triterpenoid pathway. Genes encoding the rubber biosynthetic complex, including cis-prenyltransferase (CPT), CPT-binding protein (CBP), and small rubber particle protein (SRPP), were specifically expressed in laticifers, indicating potential rubber formation. Ultrastructural and chemical analyses detect small rubber particles (similar to 150 nm) and a low-abundance cis-1,4-polyisoprene fraction in latex (0.30-0.47% w/w) with short average chain length (Mw approximate to 150-180 kDa). The streamlined repertoire of rubber biosynthetic pathway genes and the absence of Hevea-like rubber elongation factors (REFs) may be associated with the observed low rubber content and smaller molecular weight in E. lathyris. Network inference and validation identified a laticifer-specific DOF transcription factor, COGWHEEL1 (ElCOG1), that binds the promoters of ElCPT1 and butyrospermol synthase 1 (ElBUT1) and represses their activity, indicating a shared negative transcriptional control point acting on both the rubber-elongation and triterpenoid branches. In summary, this single-nucleus transcriptomic landscape of E. lathyris laticifers clarifies their metabolic specialization and establishes a framework for dissecting the regulatory programs of specialized secretory cells in latex-bearing plants.

During development, cell fate determination hinges on the dynamic activities of multiple transcription factors (TFs), but how the activities of individual TFs contribute to developmental diversity remains incompletely understood. Here, we report that BZU2/ZmMUTE is responsible for the functional and spatial heterogeneity of the four-celled stomatal complexes in maize (Zea mays), in part through its liquid-liquid phase separation (LLPS) property. Genotypic and single-cell RNA-seq analyses confirmed previous findings that ZmMUTE is associated with lineage-specific gene expression during stomatal development, while providing additional resolution of cell-type-specific transcriptional programs. The intrinsically disordered region 4 (IDR4) promotes the formation of ZmMUTE condensates, which can recruit the ZmMUTE partner ZmSCRM and associate with transcriptionally active sites. These condensates are associated with enhanced DNA binding and target gene activation, potentially influencing the development of guard cells and subsidiary cells. IDR4 is also sufficient for conferring cell-to-cell mobility to AtMUTE, highlighting its conserved role in facilitating intercellular movement. Taken together, the dynamic behavior of ZmMUTE, owing to its condensational properties, appears to orchestrate cell-type-specific regulation, thereby enabling the emergence of the unique four-celled stomatal morphology.

Secondary xylem differentiation determines wood structure and function in perennial plants, yet its regulatory mechanisms remain poorly understood in Hevea brasiliensis. Here, we generated a single-nucleus RNA sequencing (snRNA-seq) atlas from the cambium to mature xylem region in stems, resolving transcriptionally distinct cell populations corresponding to cambium, xylem mother cells, and fibre-vessel cells. Pseudotime analysis reconstructed the developmental trajectories from cambium to late xylem cell states and revealed stage-specific activation of secondary cell wall (SCW) biosynthesis programmes. Gene regulatory network analysis identified HbWRKY12a as a fibre-vessel enriched transcription factor functioning as a key regulator in SCW formation. Molecular assays demonstrated that HbWRKY12a directly binds and activates HbMYB1R1c, which subsequently modulates lignin-associated SCW deposition. Heterologous overexpression of either HbWRKY12a or HbMYB1R1c in Arabidopsis resulted in significant reductions in stem diameter and plant height. Both overexpression lines exhibited markedly decreased SCW thickness in fibre, vessels, and pith cells. These findings reveal a WRKY-MYB cascade that mediates SCW formation in H. brasiliensis, extending the classical NAC-MYB regulatory framework and providing molecular targets for improving xylem properties and stress resilience in tropical perennial crops.

Despite decades of efforts in genome sequencing and functional characterization, some important protein families remain poorly understood. In this study, we report the classification, evolution, and functions of the largely uncharacterized AIM24 protein family in plants, including the identification of a novel subfamily. We show that two AIM24 subfamilies (AIM24-A and AIM24-B) are commonly distributed in major plant groups. These two subfamilies not only have modest sequence similarities and different gene structures but also are of independent bacterial ancestry. We performed comparative functional investigations on the two AIM24 subfamilies using three model plants: the moss Physcomitrium patens, the liverwort Marchantia polymorpha, and the flowering plant Arabidopsis thaliana. Intriguingly, despite their significant differences in sequence and gene structure, both AIM24 subfamilies are involved in ER stress tolerance and the unfolded protein response (UPR). In addition, transformation of the AIM24-A gene from P. patens into the AIM24-B null mutant of A. thaliana could at least partially rescue ER stress tolerance and the UPR. We also discuss the role of AIM24 genes in plant development and other cellular activities. This study provides a unique example of parallel evolution in molecular functions and can serve as a foundation for further investigation of the AIM24 family in plants.

Seed plants have evolved mechanisms that maintain the dormancy of mature seeds until the time is appropriate for germination. Seed germination is a critical step in the plant life cycle, and it is an important trait in relation to agricultural production. The process is precisely regulated by various internal and external factors, and in particular by diverse endogenous hormones. Jasmonates (JAs) are one of the main plant hormones that mediate stress responses, and recent studies have provided evidence of their inhibitory effects on seed germination. In this review, we summarize our current understanding of the molecular mechanisms underlying the regulatory roles of JAs during the seed germination stage. We describe the crosstalk between JA and other phytohormones that influence seed germination, such as abscisic acid and gibberellic acid.

Stomata are distributed in nearly all major groups of land plants, with the only exception being liverworts. Instead of having stomata on sporophytes, many complex thalloid liverworts possess air pores in their gametophytes. At present, whether stomata in land plants are derived from a common origin remains under debate.1,2,3 In Arabidopsis thaliana, a core regulatory module for stomatal development comprises members of the bHLH transcription factor (TF) family, including AtSPCH, AtMUTE, and AtFAMA of subfamily Ia and AtSCRM1/2 of subfamily IIIb. Specifically, AtSPCH, AtMUTE, and AtFAMA each successively form heterodimers with AtSCRM1/2, which in turn regulate the entry, division, and differentiation of stomatal lineages.4,5,6,7 In the moss Physcomitrium patens, two SMF (SPCH, MUTE and FAMA) orthologs have been characterized, one of which is functionally conserved in regulating stomatal development.8,9 We here provide experimental evidence that orthologous bHLH TFs in the liverwort Marchantia polymorpha affect air pore spacing as well as the development of the epidermis and gametangiophores. We found that the bHLH Ia and IIIb heterodimeric module is highly conserved in plants. Genetic complementation experiments showed that liverwort SCRM and SMF genes weakly restored a stomata phenotype in atscrm1, atmute, and atfama mutant backgrounds in A. thaliana. In addition, homologs of stomatal development regulators FLP and MYB88 also exist in liverworts and weakly rescued the stomatal phenotype of atflp/myb88 double mutant. These results provide evidence not only for a common origin of all stomata in extant plants but also for relatively simple stomata in the ancestral plant.

How horizontal gene transfer (HGT) has contributed to the evolution of animals and plants remains a major puzzle. Despite recent progress, defining the overall scale and pattern of HGT events in land plants has been largely elusive. In this study, we performed systematic analyses for acquired genes in different plant groups and throughout land plant evolution. We found that relatively recent HGT events occurred in char-ophytes and all major land plant groups, but their frequency declined rapidly in seed plants. Two major ep -isodes of HGT events occurred in land plant evolution, corresponding to the early evolution of strepto-phytes and the origin of land plants, respectively. Importantly, a vast majority of the genes acquired in the two episodes have been retained in descendant groups, affecting numerous activities and processes of land plants. We analyzed some of the acquired genes involved in stress responses, ion and metabolite transport, growth and development, and specialized metabolism, and further assessed the cumulative ef-fects of HGT in land plants.

Natural hybridization is a crucial evolutionary process and a long-standing topic of study in evolutionary biology. Hybrid zones, where two congeneric species interact, can provide insight into the process of natural hybridization, especially with respect to how taxon diversity is maintained. In this study, we used double digest restriction-site associated DNA sequencing technology (ddRAD-seq) to examine genetic structure and estimate introgression in four hybrid zones of Ligularia tongolensis and Ligularia cymbulifera. Our analysis demonstrated that parental species were highly differentiated, whereas pairwise F-ST between parents and their hybrids was low, indicating that sympatric sites can form hybrid zones. As most F-1 hybrid individuals were observed within these zones, our finding also implied the presence of substantial barriers to interbreeding. Furthermore, some individuals that possessed the typical morphology of the parental species belonged to the F-1 generation. Genomic clines analysis revealed that a large fraction of single nucleotide polymorphisms (SNPs) deviated from a model of neutral introgression in the four hybrid zones, and most SNPs exhibited selection favoring the L. cymbulifera genotype. Bidirectional but asymmetric introgression was revealed as evident in the four hybrid zones. Habitat differences between the four hybrid zones may affect isolation barriers between both species. Taken together, these findings suggest that where incomplete reproductive barriers allow natural hybridization, the introgression between species generates rich genetic recombination that contributes to the fast adaptation and diversification of the widespread Ligularia in the Hengduan Mountains Region (HMR).

The unique morphology of grass stomata enables rapid responses to environmental changes. Deciphering the basis for these responses is critical for improving food security. We have developed a planta platform of single nucleus RNA-sequencing by combined fluorescence-activated nuclei flow sorting, and used it to identify cell types in mature and developing stomata from 33,098 nuclei of the maize epidermis. Guard and subsidiary cells displayed differential expression of genes, besides those encoding transporters, involved in the abscisic acid, CO2, Ca2+, starch metabolism, and blue light signaling pathways, implicating coordinated signal integration in speedy stomatal responses, and of genes affecting cell wall plasticity, implying a more sophisticated relationship between guard and subsidiary cells in stomatal development and dumbbell-shaped guard cell formation. The trajectory of stomatal development identified in young tissues, and by comparison to the bulk RNA-seq data of the MUTE defective mutant in stomatal development, confirmed known features and shed light on key participants in stomatal development. Our study provides a valuable, comprehensive, and fundamental foundation for further insights into grass stomatal function.

Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology.1 Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy)2 for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes.3, 4 Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of whole-genome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms.5 FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification6, 7 within organized tissues, such as roots.8, 9 The trigger to develop this virtual issue came from the publication, in 2017, of an article entitled "Guidelines for the use of flow cytometry and cell sorting in immunological studies" in the European Journal of Immunology.10 As noted in that article, one of the advantages of FCM/FCS systems is that they are relatively simple to implement, with some qualifications, which coupled with the development of user-friendly devices and software during the last 15 years led to increasing applications in other areas, such as plant sciences. However, it is also simple to implement and operate the instruments inappropriately. This calls for a comprehensive and collective summary of the best practices when applying FCM/FCS to plants, as was done for immunology. The first consideration addresses the problem that plants, particularly the vascular plants, in their commonly recognized and utilized forms, exist not as single cell suspensions (typical of immunology) but as complex three-dimensional tissues comprising cells of irregular shapes, different types and functions, that collectively cooperate to produce the final plant form. Optimal methods for producing suspensions of cells, subcellular organelles and other components appropriate for FCM/FCS from these plant tissues and organs, are therefore one of the challenges discussed in this virtual issue. We are fully aware of the mantra that "junk in equals junk out" and having samples of the highest quality prior to FCM/FCS is a critical concern we also addressed here. The second consideration relates to the vast variety of different plant species found globally, and the recognition of the consummate ability of plants to produce secondary metabolites/products, affecting DNA staining and resulting fluorescence. Again, methods for recognizing and handling the different challenges provided to FCM/FCS methods by the biochemistries of the source samples are required. The third consideration focuses on the problem of addressing the non-critical application of FCM/FCS methods developed for mammalian cell systems (typically hematopoietic) to plants without careful consideration of their appropriateness. As it will be detailed in this virtual issue, application of FCM/FCS methods to mammalian cell systems almost exclusively occurs in the context of analysis of samples that comprise a majority, often close to 100%, of single cells in suspension. For plants, particularly when using these instruments and methods for the analysis of organelles in tissue homogenates, the objects of interest comprise a very minor subpopulation of the total particles passing through the instrument. Concepts such as placing initial gates around populations defined by forward scatter (FS) versus side scatter (SS), as routinely used to define leukocytes or other mammalian cells in culture, are at best meaningless and at worst can seriously hamper proper use of the instruments to provide meaningful results. Again, plants are sources of many forms of autofluorescence; in vascular plants, chloroplasts are intensely fluorescent in the red due to the presence of chlorophyll. Phycoerythrin, a red protein-pigment complex from the light-harvesting phycobiliprotein family found in red algae and cryptophytes, is commercially employed as a fluorescent label for antibodies in cytometry. The presence of autofluorescence can restrict the wavelength bandwidths for fluorescence excitation and emission, and this can affect how best to set up FCM/FCS instruments. In order to define and enunciate best practices, we drew together a network of volunteer authors, experienced in the application of FCM/FCS to plants. We have attempted to make this network as comprehensive as possible, to allow recommendations spanning all relevant life-forms, from the simplest photosynthetic microbes, to the more complex lower and vascular plants, and encompassing also the fungi. In this endeavor, we gratefully acknowledge the support of Wiley and Attila Tarnok, EIC of Cytometry. As indicated for the Guidelines in Immunology article,9 we do wish to keep our recommendations updated. Therefore, please send us your critical comments, new ideas, practical suggestions regarding best practices, and new articles that could be useful for possible future versions of this virtual issue. To end, we would like to remember that this virtual issue reflects the vision and dream of the late Jan Suda. He has been an inspiration for all of us, and, most certainly, he left us too soon. We are sure that his legacy will persist, not only in his home country, the Czech Republic, but also across the world. We sincerely hope this virtual issue of Cytometry Part A provides an appropriate tribute. Describing how FCCS can be optimally applied to plants requires information in two general areas (a) concerning the samples being prepared and analyzed, in our case focusing on the relevant physical features of plants as organisms, and (b) concerning the instrumentation being used for this analysis, centering on sample requirements imposed by engineering design and implementation. Green plants (Viridiplantae) constitute a monophyletic clade within the tree of life and comprise oxygenic photosynthetic eukaryotes.11 The group encompasses green algae and land plants, and further splits into major clades: the Chlorophyta,12 comprising only algae, and Streptophyta formed by several algal groups (such as Zygnematophyceae and Charophyceae;,)13, 14 and the land plants (Embryophyta). Land plants further split into several groups: the possibly paraphyletic assemblage of three bryophyte lineages (Bryophyta—mosses, Marchantiophyta—liverworts, and Anthocerotophyta—hornworts) and three sequentially-splitting lineages of vascular plants: lycopods (Lycopodiophyta), ferns and horsetails (Monilophyta) and seed plants (Spermatophyta). The latter group further splits into gymnosperms (Gymnospermae; i.e., conifers, cycads, Ginkgo, and gnetophytes) and angiosperms (Angiospermae;).15-17 The current review is primarily but not exclusively focused on flow cytometric applications in flowering plants, as they represent the most diverse and economically important, and therefore best studied, group of green plants. However, we mention the other green plant lineages where necessary and we also include other organisms that are found in various parts of the Tree of Life (algae in the traditional sense, fungi) and that share certain features of body organization and life style with plants (such as complex tissues or photosynthesis), and have for a long time been a subject of Botany in the broadest sense. The life cycles of algal groups are highly variable and may comprise stages only with haplophasic (n) or diplophasic (2n) chromosome numbers, although in other species both stages are present but in separate generations.18 All land plants exhibit a characteristic life cycle which alternates between a haplophasic gametophyte and a diplophasic sporophyte. Still, the relative importance of each stage in the life cycle differs between groups: while the gametophyte stage dominates in bryophytes (and is usually the tissue that is analyzed by FCM), the sporophyte stage dominates in the vascular plant groups and is the main focus of flow cytometric investigations. Despite a significant reduction in the size of the gametophyte (comprising only up to 3–4 cells/nuclei in flowering plants), there are flow cytometric applications focused on either the independent gametophyte or the spores of ferns or on pollen grains of seed plants.19 Unlike vascular plants, fungal life cycles are mostly haplophasic, with a short (often single-celled) diplophasic stage, although most fungi (the Dikarya, i.e., the Ascomycota and the Basidiomycota) are dikaryotic (n + n) in part of their life cycles. The evolution of plant genomes is dynamic, particularly in angiosperms, encompassing a range of genomic processes including multiple rounds of whole genome duplication (polyploidization,20, 21 chromosomal rearrangements22, 23 and the turnover and evolution of repetitive DNA (including mobile elements and satellite DNA).24, 25 This is mirrored in the tremendous variation in nuclear genome sizes across green plants in general (c. 11,850-fold; 2) and flowering plants in particular (2,400-fold variation; 3,4). This has crucial implications for flow cytometric applications both with respect to technical issues (a series of internal standards of different genome size is required) and also as a study topic per se (e.g. what are the mechanisms driving genome size evolution?). Similarly, the relative content of AT versus GC base pairs is highly variable in green plants, although this variation does not strictly correlate with nuclear DNA-content (e.g., 26). While the algal groups are mostly unicellular, or comprise a rather simple multicellular thallus (e.g., Ulva, Cladophora, or Chara), land plants form complex tissues and organs. The sporophyte of vascular plants typically differentiates into roots, stems and leaves (note that the floral parts of flowering plants are derived from the leaves). Similar (yet haplophasic and thus non-homologous) structures are found in the gametophytes of bryophytes: rhizoids, cauloids, and phylloids. The specific morphology and anatomy of green plants, as distinct from other eukaryotes, naturally has multiple implications/challenges for flow cytometric analysis. Firstly, we encounter cells having thick cell walls that render flow cytometric analysis of individual cells impossible. Instead, isolated protoplast and, more commonly, nuclear suspensions are used for the analysis of plant tissues.26-28 Secondly, two types of endosymbiotic organelles, each with their own genomes, are present in most plant cells, mitochondria and plastids, and FCM applications have been designed to analyze those organelles.29-31 Lastly, plants present a wide array of chemical compounds, so-called secondary metabolites, conferring protection against factors both abiotic (e.g., UV-light, frost) and biotic (e.g., herbivores, parasites). Some of these chemical compounds (for example, tannins) directly co-interact with the DNA-binding stains used in FCM, and significantly affect the quality and reliability of such analyses.32 Flow cytometry and cytometric sorting systems are assembled from distinct engineering modules which collectively function to determine the optical properties of suspensions of biological particles, and selectively isolate these particles, or subsets thereof, for subsequent analysis and processing. The particles are typically constrained hydrodynamically within an aqueous stream to flow singly through regions of intense light, almost exclusively provided by lasers that are focused on the stream. On illumination, the particles absorb and scatter light and, if associated with fluorochromes, subsequently emit fluorescence. The intensities of the scattered and fluorescent light pulses coming from each particle are then measured. Key elements in these modules are (a) a flow cell, which spatially positions and aligns the flow stream containing the particles with the excitation light and detection axes, (b) light scatter and fluorescence detectors, screened by wavelength-appropriate filters and oriented orthogonally to the direction of the flow stream and the excitation light path, (c) electronic circuitry including analog-to-digital converters (ADCs) which convert the voltage pulses emerging from the detectors into digital values corresponding to the outputs from the individual particles, (d) computational architecture to process and store the information from these pulses for further analysis, or to use them immediately for processing sort-related decisions, and (e) mechanisms to implement individual, high-speed sorting of the particles, based on preselected combinations of optical characteristics. One of the first implementations of flow sorting, and one of the most influential, was described by Bonner et al.33 for characterization and isolation of various mammalian cell types including those of the hematopoietic system. To date, immunological applications represent the largest fraction of cytometric activities, worldwide. Most flow sorters employ a version of this original implementation, which involves precise conversion of the flow stream into a series of individual droplets, electromechanically synchronized to appear at a fixed distance below the point of laser interception (Figure 1). Based on the degree of sample dilution, some of these droplets contain the cells of interest, and can be selectively displaced into collection vessels by a process of charging the droplet at the point of its detachment from the flow stream followed by passage through a fixed electrostatic field. The rates of sorting depend on the size of the cells, which determines the size of the flow tip, and the rate of flow of the fluid stream.34 Advances in the area of instrument development have included multiplexed excitation and detection modalities to comprehensively cover the excitation and fluorescence emission spectra of the available fluorochromes.35 Recently, spectral analysis has been demonstrated as an alternative to conventional light filters in FCM.36, 37 Other advances include the use of flow tips that accommodate cells and biological particles that are larger, and sometimes much larger, than mammalian blood cells, drastic reductions in overall instrument sizes, footprints, and purchase costs, full replacement of analog by digital signal processing and the use of miniaturized fluidics systems with corresponding improvements in accuracy and reliability, and accompanied by reductions in costs of maintenance. Flow cytometry and flow cytometric sorting are not new methods. However, their use in Plant Biology has grown dramatically in the last decades, and in some cases, such as genome size measurements, these technologies have come to dominate. At the same time, instruments and associated protocols continue to be improved and expanded (e.g., bead beating, the use of tissues other than leaves, dry tissue). The literature now includes many resources outlining methods, theoretical issues, and limitations of methods (e.g., the "Flow Cytometry with Plant Cells" book,38 ESACP guidelines http://www.classimed.de/esacflow.html). However, despite this progress, it is clear from some recent publications that experimental design and manuscript review have not always kept pace with what we know about the application of FCM and cytometric sorting to plants, and this has adversely affected the quality of the results and the conclusions drawn. Contributing factors include: Examples of poor practices and erroneous theories developed as a consequence of these practices, identified by Jan Suda in the original draft, include flax genotrophs and problems with intraspecific variation reports, as reviewed in Greilhuber.35 Recent tendencies in manuscripts to justify the use of dry tissue based on existing literature frequently lack acknowledgment of necessary precautions from the prior literature. The main objective of this virtual issue is to outline key experimental issues and associated guidelines (under the heading of "Best Practices") that researchers are recommended to follow, and to provide the rationale for these recommendations, such that the guidelines may be modified with confidence as new applications emerge. We also identify those areas where the establishment of clear guidelines will require additional empirical data or theoretical work. Such guidelines will benefit researchers, facility managers, journal editors, and reviewers, since they should serve to guarantee high-quality results through elimination (or, at the very least, minimization) of artifactual variation from future research submitted for publication, as well as providing a means to identify artifacts within the published literature. The emphasis will be on providing guidelines for reviewers and for experimental design. This will NOT be a methods virtual issue in the sense of providing protocols: these are well-covered elsewhere (e.g.,27 "Flow Cytometry with Plant Cells" book,38 the supplemental material of Kron et al.,39 online resources). The authors have no conflicts of interest to declare. David Galbraith: Conceptualization; writing-original draft; writing-review and editing. João Loureiro: Conceptualization; writing-original draft; writing-review and editing. Ioanna Antoniadi: Writing-review and editing. Jillian Bainard: Writing-review and editing. Petr Bureš: Writing-review and editing. Petr Cápal: Writing-review and editing. Mariana Castro: Writing-review and editing. Sílvia Castro: Writing-review and editing. Martin Čertner: Writing-review and editing. Dora Čertnerová: Writing-review and editing. Zuzana Chumová: Writing-review and editing. Jaroslav Dolezel: Writing-review and editing. Debora Giorgi: Writing-review and editing. Brian Husband: Writing-review and editing. Filip Kolar: Writing-review and editing. Petr Koutecký: Writing-review and editing. Paul Kron: Writing-review and editing. Ilia Leitch: Writing-review and editing. Karin Ljung: Writing-review and editing. Sara Lopes: Writing-review and editing. Magdalena Lučanová: Writing-review and editing. Sergio Lucretti: Writing-review and editing. Wen Ma: Writing-review and editing. Susanne Melzer: Writing-review and editing. István Molnár: Writing-review and editing. Ondřej Novák: Writing-review and editing. Nicole Poulton: Writing-review and editing. Vladimír Skalický: Writing-review and editing. Elwira Sliwinska: Writing-review and editing. Petr Šmarda: Writing-review and editing. Tyler Smith: Writing-review and editing. Guiling Sun: Writing-review and editing. Pedro Talhinhas: Writing-review and editing. Attila Tárnok: Writing-review and editing. Eva Temsch: Writing-review and editing. Pavel Trávnícek: Writing-review and editing. Tomas Urfus: Writing-review and editing.

Increasing crop production is necessary to feed the world's expanding population, and crop breeders often utilize genetic variations to improve crop yield and quality. However, the narrow diversity of the wheat D genome seriously restricts its selective breeding. A practical solution is to exploit the genomic variations of Aegilops tauschii via introgression. Here, we established a rapid introgression platform for transferring the overall genetic variations of A. tauschii to elite wheats, thereby enriching the wheat germplasm pool. To accelerate the process, we assembled four new reference genomes, resequenced 278 accessions of A. tauschii and constructed the variation landscape of this wheat progenitor species. Genome comparisons highlighted diverse functional genes or novel haplotypes with potential applications in wheat improvement. We constructed the core germplasm of A. tauschii, including 85 accessions covering more than 99% of the species' overall genetic variations. This was crossed with elite wheat cultivars to generate an A. tauschii-wheat synthetic octoploid wheat (A-WSOW) pool. Laboratory and field analysis with two examples of the introgression lines confirmed its great potential for wheat breeding. Our high-quality reference genomes, genomic variation landscape of A. tauschii and the A-WSOW pool provide valuable resources to facilitate gene discovery and breeding in wheat.

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.

Background and aims As drought threatens the yield and quality of maize ( Zea mays L.), it is important to dissect the molecular basis of maize drought tolerance. Flavonoids, participate in the scavenging of oxygen free radicals and alleviate stress-induced oxidative damages. This study aims to dissect the function of flavonoids in the improvement of maize drought tolerance. Methods Using far-infrared imaging screening, we previously isolated a drought overly insensitivity ( doi ) mutant from an ethyl methanesulfonate (EMS)-mutagenized maize library and designated it as doi57 . In this study, we performed a physiological characterization and transcriptome profiling of doi57 in comparison to corresponding wild-type B73 under drought stress. Results Under drought stress, doi57 seedlings displayed lower leaf-surface temperature (LST), faster water loss, and better performance in growth than B73. Transcriptome analysis reveals that key genes involved in flavonoid biosynthesis are enriched among differentially expressed genes in doi57 . In line with these results, more flavonols and less hydrogen peroxide (H 2 O 2 ) were accumulated in guard cells of doi57 than in those of B73 with the decrease of soil water content (SWC). Moreover, the capacity determined from doi57 seedling extracts to scavenge oxygen free radicals was more effective than that of B73 under the drought treatment. Additionally, doi57 seedlings had higher photosynthetic rates, stomatal conductance, transpiration rates, and water use efficiency than B73 exposed to drought stress, resulting in high biomass and greater root/shoot ratios in doi57 mutant plants. Conclusion Flavonoids may facilitate maize seedling drought tolerance by lowering drought-induced oxidative damage as well regulating stomatal movement.

Paulownias are among the fastest growing trees in the world, but they often suffer tremendous loss of wood production due to infection by Paulownia witches' broom (PaWB) phytoplasmas. In this study, we have sequenced and assembled a high-quality nuclear genome of Paulownia fortunei, a commonly cultivated paulownia species. The assembled genome of P. fortunei is 511.6 Mb in size, with 93.2% of its sequences anchored to 20 pseudo-chromosomes, and it contains 31 985 protein-coding genes. Phylogenomic analyses show that the family Paulowniaceae is sister to a clade composed of Phrymaceae and Orobanchaceae. Higher photosynthetic efficiency is achieved by integrating C3 photosynthesis and the crassulacean acid metabolism pathway, which may contribute to the extremely fast growth habit of paulownia trees. Comparative transcriptome analyses reveal modules related to cambial growth and development, photosynthesis, and defense responses. Additional genome sequencing of PaWB phytoplasma, combined with functional analyses, indicates that the effector PaWB-SAP54 interacts directly with Paulownia PfSPLa, which in turn causes the degradation of PfSPLa by the ubiquitin-mediated pathway and leads to the formation of witches' broom. Taken together, these results provide significant insights into the biology of paulownias and the regulatory mechanism for the formation of PaWB.

Land plants have been intimately associated with fungi over the course of their evolution. Because of their lack of sophisticated protective structures, early land plants would conceivably have required additional defense strategies against microbial pathogens, including various fungi. On the other hand, a symbiotic association between plants and fungi is instrumental to plant adaptation to terrestrial environments (Selosse & Le Tacon, 1998; Bidartondo et al., 2011; Martin et al., 2017).

Natural hybridization is a common key evolutionary process and a long-standing topic in the plant taxon. Hybrid zones, where two closely related species interact, can provide an avenue to insight the process, illuminating the maintenance of the taxon diversity. Incomplete isolation barriers between Ligularia species generally form hybrid zones. In this study, we used the double digest restriction-site associated DNA sequencing technology (ddRAD-seq) to examine genetic structure and estimate introgression in four newly discovered hybrid zones between L. tongolensis and L. cymbulifera. Our results showed high differentiation between parental species, whereas pairwise FST between parents and their hybrids was low, further corroborating sympatric site form hybrid zones. Moreover, most F1 hybrid individuals were observed within the four hybrid zones implying the presence of substantial barriers to interbreeding. An analysis of genomic clines indicated that a large fraction of loci deviated from a model of neutral introgression in the four hybrid zones, of which most loci exhibited selection favouring L. cymbulifera genotypes. Our analysis demonstrated bidirectional but asymmetric introgression appearing in the four hybrid zones. The different habitats among four hybrid zones may affect isolation barriers between both species. Natural hybridization with post-zygotic isolation barriers may significantly contribute to the diversification of Ligularia in the HMR.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The P. binpinnatifidus complex included most of the Panax species distributed in Sino-Himalaya regions except for P. pseudoginseng, P. stipuleanatus and P. notoginseng. However, the delimitation and identification of these taxa within the species complex are very difficult due to the existence of morphological intermediates, and their evolutionary relationships remain unresolved despite several studies have been carried out based on traditional DNA markers. The taxonomic uncertainty hinders the identification, conservation and exploration of these wild populations of Panax. To study this species complex, we employed ddRAD-seq data of these taxa from 18 different localities of southwestern China, using two RAD analysis pipelines, STACKS and pyRAD. Based on the results of phylogenetic analysis, the species complex was divided into four clades with high supports, which largely agreed with morphologically described species. Two clades, corresponding to P. vietnamensis and P. zingiberensis, respectively, were sister groups, indicating that these two species had a closer genetic relationship; the third clade was consisted of samples with bamboo-like rhizomes named as P. wangianus clade, and the fourth one with moniliform rhizomes was named as P. bipinnatifidus clade. The population genetic structure analysis and D-statistics test showed the localized admixture among these species, which indicated that introgression had occurred among the related lineages continuously distributed in southeastern Yunnan and adjacent regions.