Streamlined spatial and environmental expression signatures characterize the minimalist duckweed Wolffia australiana.

Single-cell genomics permits a new resolution in the examination of molecular and cellular dynamics, allowing global, parallel assessments of cell types and cellular behaviors through development and in response to environmental circumstances, such as interaction with water and the light-dark cycle of the Earth. Here, we leverage the smallest, and possibly most structurally reduced plant, the semi-aquatic Wolffia australiana to understand dynamics of cell expression in these contexts at the whole plant level. We examined single cell resolution RNA sequencing data, and found Wolffia cells divide into four principal clusters representing the above and below water-situated parenchyma and epidermis. While these tissues share transcriptomic similarity with model plants, they display distinct adaptations that Wolffia has made for the aquatic environment. Within this broad classification, discrete subspecializations are evident with select cells showing unique transcriptomic signatures associated with developmental maturation and specialized physiologies. Assessing this simplified biological system temporally at two key time-of-day (TOD) transitions, we identify additional TOD-responsive genes previously overlooked in whole plant transcriptomic approaches and demonstrate that the core circadian clock machinery and its downstream responses can vary in cell-specific manners, even in this simplified system. Distinctions between cell types and their responses to submergence and/or TOD are driven by expression changes of unexpectedly few genes, characterizing Wolffia as a highly streamlined organism with the majority of genes dedicated to fundamental cellular processes. Wolffia provides a unique opportunity to apply reductionist biology to elucidate signaling functions at the organismal level, for which this work provides a powerful resource.

A diffusible small-RNA-based Turing system dynamically coordinates organ polarity.

The formation of a flat and thin leaf presents a developmentally challenging problem, requiring intricate regulation of adaxial-abaxial (top-bottom) polarity. The patterning principles controlling the spatial arrangement of these domains during organ growth have remained unclear. Here we show that this regulation in Arabidopsis thaliana is achieved by an organ-autonomous Turing reaction-diffusion system centred on mobile small RNAs. The data illustrate how Turing dynamics transiently instructed by prepatterned information is sufficient to self-sustain properly oriented polarity in a dynamic, growing organ, presenting intriguing parallels to left-right patterning in the vertebrate embryo. Computational modelling demonstrates that this self-organizing system continuously adapts to coordinate the robust planar polarity of a flat leaf while affording flexibility to generate the tissue patterns of evolutionarily diverse organ shapes. Our findings identify a small-RNA-based Turing network as a dynamic regulator of organ polarity that accounts for leaf shape diversity at the level of the individual organ, plant or species.

microRNA165 and 166 modulate response of the Arabidopsis root apical meristem to salt stress.

In plants, developmental plasticity allows for the modulation of organ growth in response to environmental cues. Being in contact with soil, roots are the first organ that responds to various types of soil abiotic stress such as high salt concentration. In the root, developmental plasticity relies on changes in the activity of the apical meristem, the region at the tip of the root where a set of self-renewing undifferentiated stem cells sustain growth. Here, we show that salt stress promotes differentiation of root meristem cells via reducing the dosage of the microRNAs miR165 and 166. By means of genetic, molecular and computational analysis, we show that the levels of miR165 and 166 respond to high salt concentration, and that miR165 and 166-dependent PHABULOSA (PHB) modulation is central to the response of root growth to this stress. Specifically, we show that salt-dependent reduction of miR165 and 166 causes a rapid increase in PHB expression and, hence, production of the root meristem pro-differentiation hormone cytokinin. Our data provide direct evidence for how the miRNA-dependent modulation of transcription factor dosage mediates plastic development in plants.

The START domain potentiates HD-ZIPIII transcriptional activity.

The CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) transcription factors (TFs) were repeatedly deployed over 725 million years of evolution to regulate central developmental innovations. The START domain of this pivotal class of developmental regulators was recognized over 20 years ago, but its putative ligands and functional contributions remain unknown. Here, we demonstrate that the START domain promotes HD-ZIPIII TF homodimerization and increases transcriptional potency. Effects on transcriptional output can be ported onto heterologous TFs, consistent with principles of evolution via domain capture. We also show the START domain binds several species of phospholipids, and that mutations in conserved residues perturbing ligand binding and/or its downstream conformational readout abolish HD-ZIPIII DNA-binding competence. Our data present a model in which the START domain potentiates transcriptional activity and uses ligand-induced conformational change to render HD-ZIPIII dimers competent to bind DNA. These findings resolve a long-standing mystery in plant development and highlight the flexible and diverse regulatory potential coded within this widely distributed evolutionary module.

Specification of leaf dorsiventrality via a prepatterned binary readout of a uniform auxin input.

Developmental boundaries play an important role in coordinating the growth and patterning of lateral organs. In plants, specification of dorsiventrality is critical to leaf morphogenesis. Despite its central importance, the mechanism by which leaf primordia acquire adaxial versus abaxial cell fates to establish dorsiventrality remains a topic of much debate. Here, by combining time-lapse confocal imaging, cell lineage tracing and molecular genetic analyses, we demonstrate that a stable boundary between adaxial and abaxial cell fates is specified several plastochrons before primordium emergence when high auxin levels accumulate on a meristem prepattern formed by the AS2 and KAN1 transcription factors. This occurrence triggers a transient induction of ARF3 and an auxin transcriptional response in AS2-marked progenitors that distinguishes adaxial from abaxial identity. As the primordium emerges, dynamic shifts in auxin distribution and auxin-related gene expression gradually resolve this initial polarity into the stable regulatory network known to maintain adaxial-abaxial polarity within the developing organ. Our data show that spatial information from an AS2-KAN1 meristem prepattern governs the conversion of a uniform auxin input into an ARF-dependent binary auxin response output to specify adaxial-abaxial polarity. Auxin thus serves as a single morphogenic signal that orchestrates distinct, spatially separated responses to coordinate the positioning and emergence of a new organ with its patterning.

Crafting a blueprint for single-cell RNA sequencing.

Droplet-based single-cell RNA sequencing (scRNA-Seq) has moved rapidly from a technology with great potential to a method applied to ever-broader questions. The detailed information that scRNA-Seq offers has proven incredibly powerful in resolving cell responses to developmental and environmental cues. However, to maximize the potential of this technology, a panoply of upstream, practical points require consideration. Principal among these are the optimization of cell-isolation procedures, accommodating biotic/abiotic stress responses, and discerning the number of cells and sequencing reads needed. To complement excellent reviews outlining applications and data analysis tools for scRNA-Seq, we here discuss these considerations and provide practical tips to tailor experimental design and ensure the best possible outcome.

Sponging of glutamate at the outer plasma membrane surface reveals roles for glutamate in development.

Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals, and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center, number of lateral roots, vegetative growth, and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.

Distinct identities of leaf phloem cells revealed by single cell transcriptomics.

The leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about leaf vascular cells, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading, yet PP has been characterized only microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified at least 19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. We provide evidence that PP development occurs independently from ALTERED PHLOEM DEVELOPMENT, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CCs), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our single-cell RNA-sequencing analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.

Genome-wide analysis of plant miRNA action clarifies levels of regulatory dynamics across developmental contexts.

Development of complex organisms requires the delicate and dynamic spatiotemporal regulation of gene expression. Central to this are microRNAs (miRNAs). These mobile small RNAs offer specificity in conveying positional information and versatility in patterning the outcomes of gene expression. However, the parameters that shape miRNA output during development are still to be clarified. Here, we address this question on a genome-wide scale, using the maize shoot apex as a model. We show that patterns and levels of miRNA accumulation are largely determined at the transcriptional level, but are finessed post-transcriptionally in a tissue-dependent manner. The stem cell environments of the shoot apical meristem and vasculature appear particularly liable to this. Tissue-specific effects are also apparent at the level of target repression, with target cleavage products in the vasculature exceeding those of other tissues. Our results argue against a clearance mode of regulation purely at the level of transcript cleavage, leading us to propose that transcript cleavage provides a baseline level of target repression, onto which miRNA-driven translational repression can act to toggle the mode of target regulation between clearance and rheostat. Our data show how the inherent complexities of miRNA pathways allow the accumulation and activity of these small RNAs to be tailored in space and time to bring about the gene expression versatility needed during development.

To move or not to move: roles and specificity of plant RNA mobility.

Intercellular communication in plants coordinates cellular functions during growth and development, and in response to environmental cues. RNAs figure prominently among the mobile signaling molecules used. Many hundreds of RNA species move over short and long distances, and can be mutually exchanged in biotic interactions. Understanding the specificity determinants of RNA mobility and the physiological relevance of this phenomenon are areas of active research. Here, we highlight the recent progress in our knowledge of small RNA and messenger RNA movement. Particular emphasis is given to novel insight into the specificity determinants of messenger RNA mobility, the role of small RNA movement in development, and the specificity of RNA exchange in plant-plant and plant-microbe interactions.

Genomic prediction of maize microphenotypes provides insights for optimizing selection and mining diversity.

Effective evaluation of millions of crop genetic stocks is an essential component of exploiting genetic diversity to achieve global food security. By leveraging genomics and data analytics, genomic prediction is a promising strategy to efficiently explore the potential of these gene banks by starting with phenotyping a small designed subset. Reliable genomic predictions have enhanced selection of many macroscopic phenotypes in plants and animals. However, the use of genomicprediction strategies for analysis of microscopic phenotypes is limited. Here, we exploited the power of genomic prediction for eight maize traits related to the shoot apical meristem (SAM), the microscopic stem cell niche that generates all the above-ground organs of the plant. With 435 713 genomewide single-nucleotide polymorphisms (SNPs), we predicted SAM morphology traits for 2687 diverse maize inbreds based on a model trained from 369 inbreds. An empirical validation experiment with 488 inbreds obtained a prediction accuracy of 0.37-0.57 across eight traits. In addition, we show that a significantly higher prediction accuracy was achieved by leveraging the U value (upper bound for reliability) that quantifies the genomic relationships of the validation set with the training set. Our findings suggest that double selection considering both prediction and reliability can be implemented in choosing selection candidates for phenotyping when exploring new diversity is desired. In this case, individuals with less extreme predicted values and moderate reliability values can be considered. Our study expands the turbocharging gene banks via genomic prediction from the macrophenotypes into the microphenotypic space.

Small RNAs as plant morphogens.

The coordination of cell fate decisions within complex multicellular structures rests on intercellular communication. To generate ordered patterns, cells need to know their relative positions within the growing structure. This is commonly achieved via the production and perception of mobile signaling molecules. In animal systems, such positional signals often act as morphogens and subdivide a field of cells into domains of discrete cell identities using a threshold-based readout of their mobility gradient. Reflecting the independent origin of multicellularity, plants evolved distinct signaling mechanisms to drive cell fate decisions. Many of the basic principles underlying developmental patterning are, however, shared between animals and plants, including the use of signaling gradients to provide positional information. In plant development, small RNAs can act as mobile instructive signals, and similar to classical morphogens in animals, employ a threshold-based readout of their mobility gradient to generate precisely defined cell fate boundaries. Given the distinctive nature of peptide morphogens and small RNAs, how might mechanisms underlying the function of traditionally morphogens be adapted to create morphogen-like behavior using small RNAs? In this review, we highlight the contributions of mobile small RNAs to pattern formation in plants and summarize recent studies that have advanced our understanding regarding the formation, stability, and interpretation of small RNA gradients.

A high-resolution gene expression atlas links dedicated meristem genes to key architectural traits.

The shoot apical meristem (SAM) orchestrates the balance between stem cell proliferation and organ initiation essential for postembryonic shoot growth. Meristems show a striking diversity in shape and size. How this morphological diversity relates to variation in plant architecture and the molecular circuitries driving it are unclear. By generating a high-resolution gene expression atlas of the vegetative maize shoot apex, we show here that distinct sets of genes govern the regulation and identity of stem cells in maize versus Arabidopsis. Cell identities in the maize SAM reflect the combinatorial activity of transcription factors (TFs) that drive the preferential, differential expression of individual members within gene families functioning in a plethora of cellular processes. Subfunctionalization thus emerges as a fundamental feature underlying cell identity. Moreover, we show that adult plant characters are, to a significant degree, regulated by gene circuitries acting in the SAM, with natural variation modulating agronomically important architectural traits enriched specifically near dynamically expressed SAM genes and the TFs that regulate them. Besides unique mechanisms of maize stem cell regulation, our atlas thus identifies key new targets for crop improvement.

Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single-Cell RNA Sequencing.

High-throughput single-cell RNA sequencing (scRNA-seq) is becoming a cornerstone of developmental research, providing unprecedented power in understanding dynamic processes. Here, we present a high-resolution scRNA-seq expression atlas of the Arabidopsis root composed of thousands of independently profiled cells. This atlas provides detailed spatiotemporal information, identifying defining expression features for all major cell types, including the scarce cells of the quiescent center. These reveal key developmental regulators and downstream genes that translate cell fate into distinctive cell shapes and functions. Developmental trajectories derived from pseudotime analysis depict a finely resolved cascade of cell progressions from the niche through differentiation that are supported by mirroring expression waves of highly interconnected transcription factors. This study demonstrates the power of applying scRNA-seq to plants and provides an unparalleled spatiotemporal perspective of root cell differentiation.

In Situ Localization of Small RNAs in Plants.

Small RNAs have vital roles in numerous aspects of plant biology. Deciphering their precise contributions requires knowledge of a small RNA's spatiotemporal pattern of accumulation. The in situ hybridization protocol described here takes advantage of locked nucleic acid (LNA) oligonucleotide probes to visualize small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within 2-6 days.

Intragenic Meiotic Crossovers Generate Novel Alleles with Transgressive Expression Levels.

Meiotic recombination is an evolutionary force that generates new genetic diversity upon which selection can act. Whereas multiple studies have assessed genome-wide patterns of recombination and specific cases of intragenic recombination, few studies have assessed intragenic recombination genome-wide in higher eukaryotes. We identified recombination events within or near genes in a population of maize recombinant inbred lines (RILs) using RNA-sequencing data. Our results are consistent with case studies that have shown that intragenic crossovers cluster at the 5' ends of some genes. Further, we identified cases of intragenic crossovers that generate transgressive transcript accumulation patterns, that is, recombinant alleles displayed higher or lower levels of expression than did nonrecombinant alleles in any of ∼100 RILs, implicating intragenic recombination in the generation of new variants upon which selection can act. Thousands of apparent gene conversion events were identified, allowing us to estimate the genome-wide rate of gene conversion at SNP sites (4.9 × 10-5). The density of syntenic genes (i.e., those conserved at the same genomic locations since the divergence of maize and sorghum) exhibits a substantial correlation with crossover frequency, whereas the density of nonsyntenic genes (i.e., those which have transposed or been lost subsequent to the divergence of maize and sorghum) shows little correlation, suggesting that crossovers occur at higher rates in syntenic genes than in nonsyntenic genes. Increased rates of crossovers in syntenic genes could be either a consequence of the evolutionary conservation of synteny or a biological process that helps to maintain synteny.