Root Cell Types Research Articles

PLETHORA‐WOX5 interaction and subnuclear localization control Arabidopsis root stem cell maintenance

Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL‐RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion‐like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT‐WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination.

Single-cell RNA sequencing reveals the landscape of maize root tips and assists in identification of cell type-specific nitrate-response genes

The root system is fundamental for maize growth and yield. Characterizing its heterogeneity and cell type-specific response to nitrate at the single-cell level will shed light on root development and nutrient uptake. We profiled the transcriptomes of >7000 cells derived from root tips of maize seedlings grown on media with or without nitrate, and identified 11 major cell types or tissues and 85 cell type-specific nitrate-response genes, including several known nitrate metabolic genes. A pseudotime analysis showed a continuous pseudotime series with the beginning at meristematic zone cells and showed that the root hair cell was derived by differentiation of a subset of epidermal cells. Interspecies comparison of root cells between maize and rice revealed the conservation and divergence of the root cell types and identified 57, 216, and 80 conserved orthologous genes in root hair, endodermis, and phloem cells respectively. This study provides a global view of maize root tip developmental processes and responses to nitrate at the single-cell level. The genes described in the present work could serve as targets for further genetic analyses and accurate regulation of gene expression and phenotypic variation in specific cell types or tissues.

A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants.

SummaryIn all multicellular organisms, transcriptional networks orchestrate organ development. The Arabidopsis root, with its simple structure and indeterminate growth, is an ideal model to investigate the spatiotemporal transcriptional signatures underlying developmental trajectories. To map gene expression dynamics across root cell types and developmental time, we built a comprehensive, organ-scale atlas at single cell resolution. In addition to estimating developmental progressions in pseudotime, we employed the mathematical concept of optimal transport to infer developmental trajectories and identify their underlying regulators. To demonstrate the utility of the atlas to interpret new datasets, we profiled mutants for two key transcriptional regulators at single cell resolution, shortroot and scarecrow. We report transcriptomic and in vivo evidence for tissue trans-differentiation underlying a mixed cell identity phenotype in scarecrow. Our results support the atlas as a rich community resource for unraveling the transcriptional programs that specify and maintain cell identity to regulate spatiotemporal organ development.

Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate

Nitrate is a nutrient and a potent signal that impacts global gene expression in plants. However, the regulatory factors controlling temporal and cell type-specific nitrate responses remain largely unknown. We assayed nitrate-responsive transcriptome changes in five major root cell types of the Arabidopsis thaliana root as a function of time. We found that gene-expression response to nitrate is dynamic and highly localized and predicted cell type-specific transcription factor (TF)-target interactions. Among cell types, the endodermis stands out as having the largest and most connected nitrate-regulatory gene network. ABF2 and ABF3 are major hubs for transcriptional responses in the endodermis cell layer. We experimentally validated TF-target interactions for ABF2 and ABF3 by chromatin immunoprecipitation followed by sequencing and a cell-based system to detect TF regulation genome-wide. Validated targets of ABF2 and ABF3 account for more than 50% of the nitrate-responsive transcriptome in the endodermis. Moreover, ABF2 and ABF3 are involved in nitrate-induced lateral root growth. Our approach offers an unprecedented spatiotemporal resolution of the root response to nitrate and identifies important components of cell-specific gene regulatory networks.

Potential Networks of Nitrogen-Phosphorus-Potassium Channels and Transporters in Arabidopsis Roots at a Single Cell Resolution.

Nitrogen (N), phosphorus (P), and potassium (K) are three major macronutrients essential for plant life. These nutrients are acquired and transported by several large families of transporters expressed in plant roots. However, it remains largely unknown how these transporters are distributed in different cell-types that work together to transfer the nutrients from the soil to different layers of root cells and eventually reach vasculature for massive flow. Using the single cell transcriptomics data from Arabidopsis roots, we profiled the transcriptional patterns of putative nutrient transporters in different root cell-types. Such analyses identified a number of uncharacterized NPK transporters expressed in the root epidermis to mediate NPK uptake and distribution to the adjacent cells. Some transport genes showed cortex- and endodermis-specific expression to direct the nutrient flow toward the vasculature. For long-distance transport, a variety of transporters were shown to express and potentially function in the xylem and phloem. In the context of subcellular distribution of mineral nutrients, the NPK transporters at subcellular compartments were often found to show ubiquitous expression patterns, which suggests function in house-keeping processes. Overall, these single cell transcriptomic analyses provide working models of nutrient transport from the epidermis across the cortex to the vasculature, which can be further tested experimentally in the future.

Innovation, conservation, and repurposing of gene function in root cell type development

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Gene Regulatory Networks Shape Developmental Plasticity of Root Cell Types Under Water Extremes

Understanding how roots modulate development under varied irrigation or rainfall is crucial for development of climate resilient crops. We established a toolbox of tagged rice lines to profile translating mRNAs and chromatin accessibility within specific cell populations. We used these to study roots in a range of environments: plates in the lab, controlled greenhouse stress and recovery conditions, and outdoors in a paddy. Integration of chromatin and mRNA data resolves regulatory networks of: cycle genes in proliferating cells that attenuate DNA synthesis under submergence; genes involved in auxin signaling, the circadian clock, and small RNA regulation in ground tissue; and suberin biosynthesis, iron transporters, and nitrogen assimilation in endodermal/exodermal cells modulated with water availability. By applying a systems approach we identify known and candidate driver transcription factors of water deficit responses and xylem development plasticity. Collectively, this resource will facilitate genetic improvements in root systems for optimal climate resilience.

Expression Partitioning of Duplicate Genes at Single Cell Resolution in Arabidopsis Roots.

Gene duplication is a key evolutionary phenomenon, prevalent in all organisms but particularly so in plants, where whole genome duplication (WGD; polyploidy) is a major force in genome evolution. Much effort has been expended in attempting to understand the evolution of duplicate genes, addressing such questions as why some paralog pairs rapidly return to single copy status whereas, in other pairs, both paralogs are retained and may diverge in expression pattern or function. The effect of a gene – its site of expression and thus the initial locus of its function – occurs at the level of a cell comprising a single cell type at a given state of the cell’s development. Using Arabidopsis thaliana single cell transcriptomic data we categorized patterns of expression for 11,470 duplicate gene pairs across 36 cell clusters comprising nine cell types and their developmental states. Among these 11,470 pairs, 10,187 (88.8%) had at least one copy expressed in at least one of the 36 cell clusters. Pairs produced by WGD more often had both paralogs expressed in root cells than did pairs produced by small scale duplications. Three quarters of gene pairs expressed in the 36 cell clusters (7,608/10,187) showed extreme expression bias in at least one cluster, including 352 cases of reciprocal bias, a pattern consistent with expression subfunctionalization. More than twice as many pairs showed reciprocal expression bias between cell states than between cell types or between roots and leaves. A group of 33 gene pairs with reciprocal expression bias showed evidence of concerted divergence of gene networks in stele vs. epidermis. Pairs with both paralogs expressed without bias were less likely to have paralogs with divergent mutant phenotypes; such bias-free pairs showed evidence of preservation by maintenance of dosage balance. Overall, we found considerable evidence of shifts in gene expression following duplication, including in >80% of pairs encoding 7,653 genes expressed ubiquitously in all root cell types and states for which we inferred the polarity of change.

Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3.

Cytoplasmic calcium ([Ca2+]cyt) is a well-characterized second messenger in eukaryotic cells. An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation. As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained. However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq. TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements.

Plant cells can change their identity based on positional information, a mechanism that confers developmental plasticity to plants. This ability, common to distinct multicellular organisms, is particularly relevant for plant phloem cells. Protophloem sieve elements (PSEs), one type of phloem conductive cells, act as the main organizers of the phloem pole, which comprises four distinct cell files organized in a conserved pattern. Here, we report how Arabidopsis roots generate a reservoir of meristematic phloem cells competent to swap their cell identities. Although PSE misspecification induces cell identity hybridism, the activity of RECEPTOR LIKE PROTEIN KINASE 2 (RPK2) by perceiving CLE45 peptide contributes to restrict PSE identity to the PSE position. By maintaining a spatiotemporal window when PSE and PSE-adjacent cells' identities are interchangeable, CLE45 signaling endows phloem cells with the competence to re-pattern a functional phloem pole when protophloem fails to form.

Innovation, Conservation and Repurposing of Gene Function in Plant Root Cell Type Development

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato and Arabidopsis tissues suggest increased expression conservation of root meristems compared with other homologous tissues. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher-order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Comparative proteome and metabolome analyses of latex-exuding and non-exuding Taraxacum koksaghyz roots provide insights into laticifer biology.

Taraxacum koksaghyz has been identified as one of the most promising alternative rubber crops. Its high-quality rubber is produced in the latex of laticifers, a specialized cell type that is organized in a network of elongated tubules throughout the entire plant body. In order to gain insights into the physiological role(s) of latex and hence laticifer biology, we examine the effects of barnase-induced latex RNA degradation on the metabolite and protein compositions in the roots. We established high-quality datasets that enabled precise discrimination between cellular and physiological processes in laticifers and non-laticifer cell types of roots at different vegetative stages. We identified numerous latex-specific proteins, including a perilipin-like protein that has not been studied in plants yet. The barnase-expressing plants revealed a phenotype that did not exude latex, which may provide a valuable genetic basis for future studies of plant-environment interactions concerning latex and also help to clarify the evolution and arbitrary distribution of latex throughout the plant kingdom. The overview of temporal changes in composition and protein abundance provided by our data opens the way for a deeper understanding of the molecular interactions, reactions, and network relationships that underlie the different metabolic pathways in the roots of this potential rubber crop.

Cis-Regulatory Code for Predicting Plant Cell-Type Transcriptional Response to High Salinity.

Multicellular organisms have diverse cell types with distinct roles in development and responses to the environment. At the transcriptional level, the differences in the environmental response between cell types are due to differences in regulatory programs. In plants, although cell-type environmental responses have been examined, it is unclear how these responses are regulated. Here, we identify a set of putative cis-regulatory elements (pCREs) enriched in the promoters of genes responsive to high-salinity stress in six Arabidopsis (Arabidopsis thaliana) root cell types. We then use these pCREs to establish cis-regulatory codes (i.e. models predicting whether a gene is responsive to high salinity for each cell type with machine learning). These pCRE-based models outperform models using in vitro binding data of 758 Arabidopsis transcription factors. Surprisingly, organ pCREs identified based on the whole-root high-salinity response can predict cell-type responses as well as pCREs derived from cell-type data, because organ and cell-type pCREs predict complementary subsets of high-salinity response genes. Our findings not only advance our understanding of the regulatory mechanisms of the plant spatial transcriptional response through cis-regulatory codes but also suggest broad applicability of the approach to any species, particularly those with little or no trans-regulatory data.

Drought stress triggers the accumulation of NO and SNOs in cortical cells of Lotus japonicus L. roots and the nitration of proteins with relevant metabolic function

Drought is considered one of the abiotic stresses with significant implications on plant productivity. Previously, we have shown that water deficit produces a differential nitro-oxidative stress in roots and leaves of Lotus japonicus L. plants. Using this model legume, we studied the nitro-oxidative stress in drought-stressed roots by complementary biochemical, cellular and proteomic approaches. Cellular analyses of root cross-sections by confocal laser scanning microscopy (CLSM) using specific fluorescent probes for superoxide radical (O2−), nitric oxide (NO), peroxynitrite (ONOO−) and S-nitrosothiols (SNOs) showed that drought stress causes a differential cellular localization of these reactive species. Mainly, O2− and ONOO− had a wide distribution in almost all root cell types (xylem, parenchyma, and peridermis), whereas NO and SNOs accumulated in cortical cells (peridermis). Liquid chromatography-electrospray/mass spectrometry (LC-ES/MS) analyses showed that the content of ascorbate, S-nitrosoglutaathione (GSNO), and reduced glutathione (GSH) in drought-stressed roots was drastically diminished. Nitroproteome analysis by two-dimensional gel electrophoresis and mass spectrometry allowed to identify 13 tyrosine-nitrated proteins such as methionine synthase, Hsp70, adenosyl-homocysteinase, peroxidase, alcohol dehydrogenases, glutamine synthetase, fructokinase, 1,3-beta-glucanase, chitinases, endochitinase, among others which are directly (24%) or indirectly (74%) related to plant defense. Taken together, these results indicate that drought-stressed roots have an active metabolism of reactive oxygen and nitrogen species (ROS and RNS) characterized by an increase of protein nitration and accumulation of NO and SNOs in cortical cells. The possibility of autophagy taking place in the stressed roots is also discussed.

Minimum requirements for changing and maintaining endodermis cell identity in the Arabidopsis root.

Changes in gene regulation during differentiation are governed by networks of transcription factors. The Arabidopsis root endodermis is a tractable model to address how transcription factors contribute to differentiation. We used a bottom-up approach to understand the extent to which transcription factors required for endodermis differentiation can confer endodermis identity to a non-native cell-type. Our results show the transcription factors SHORTROOT and MYB36 alone have limited ability to induce ectopic endodermal features in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced endodermis identity acquisition. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer, which has a transcriptional signature similar to the endodermis. These results demonstrate other root cell-types can be forced to differentiate into endodermis and highlights a previously unappreciated role for receptor-kinase signaling in maintaining endodermis identity.

Integration of large-scale data for extraction of integrated Arabidopsis root cell-type specific models

Plant organs consist of multiple cell types that do not operate in isolation, but communicate with each other to maintain proper functions. Here, we extract models specific to three developmental stages of eight root cell types or tissue layers in Arabidopsis thaliana based on a state-of-the-art constraint-based modeling approach with all publicly available transcriptomics and metabolomics data from this system to date. We integrate these models into a multi-cell root model which we investigate with respect to network structure, distribution of fluxes, and concordance to transcriptomics and proteomics data. From a methodological point, we show that the coupling of tissue-specific models in a multi-tissue model yields a higher specificity of the interconnected models with respect to network structure and flux distributions. We use the extracted models to predict and investigate the flux of the growth hormone indole-3-actetate and its antagonist, trans-Zeatin, through the root. While some of predictions are in line with experimental evidence, constraints other than those coming from the metabolic level may be necessary to replicate the flow of indole-3-actetate from other simulation studies. Therefore, our work provides the means for data-driven multi-tissue metabolic model extraction of other Arabidopsis organs in the constraint-based modeling framework.

Quantitative 3D imaging of cell level auxin and cytokinin response ratios in soybean roots and nodules.

Legume-Rhizobium symbiosis results in root nodules where rhizobia fix atmospheric nitrogen into plant usable forms in exchange for plant-derived carbohydrates. The development of these specialized root organs involves a set of carefully orchestrated plant hormone signalling. In particular, a spatio-temporal balance between auxin and cytokinin appears to be crucial for proper nodule development. We put together a construct that carried nuclear localized fluorescence sensors for auxin and cytokinin and used two photon induced fluorescence microscopy for concurrent quantitative 3-dimensional imaging to determine cellular level auxin and cytokinin outputs and ratios in root and nodule tissues of soybean. The use of nuclear localization signals on the markers and nuclei segmentation during image processing enabled accurate monitoring of outputs in 3D image volumes. The ratiometric method used here largely compensates for variations in individual outputs due to sample turbidity and scattering, an inherent issue when imaging thick root and nodule samples typical of many legumes. Overlays of determined auxin/cytokinin ratios on specific root zones and cell types accurately reflected those predicted based on previously reported outputs for each hormone individually. Importantly, distinct auxin/cytokinin ratios corresponded to distinct nodule cell types indicating a key role for these hormones in nodule cell type identity.

A dynamic genetic-hormonal regulatory network model explains multiple cellular behaviors of the root apical meristem of Arabidopsis thaliana.

The study of the concerted action of hormones and transcription factors is fundamental to understand cell differentiation and pattern formation during organ development. The root apical meristem of Arabidopsis thaliana is a useful model to address this. It has a stem cell niche near its tip conformed of a quiescent organizer and stem or initial cells around it, then a proliferation domain followed by a transition domain, where cells diminish division rate before transiting to the elongation zone; here, cells grow anisotropically prior to their final differentiation towards the plant base. A minimal model of the gene regulatory network that underlies cell-fate specification and patterning at the root stem cell niche was proposed before. In this study, we update and couple such network with both the auxin and cytokinin hormone signaling pathways to address how they collectively give rise to attractors that correspond to the genetic and hormonal activity profiles that are characteristic of different cell types along A. thaliana root apical meristem. We used a Boolean model of the genetic-hormonal regulatory network to integrate known and predicted regulatory interactions into alternative models. Our analyses show that, after adding some putative missing interactions, the model includes the necessary and sufficient components and regulatory interactions to recover attractors characteristic of the root cell types, including the auxin and cytokinin activity profiles that correlate with different cellular behaviors along the root apical meristem. Furthermore, the model predicts the existence of activity configurations that could correspond to the transition domain. The model also provides a possible explanation for apparently paradoxical cellular behaviors in the root meristem. For example, how auxin may induce and at the same time inhibit WOX5 expression. According to the model proposed here the hormonal regulation of WOX5 might depend on the cell type. Our results illustrate how non-linear multi-stable qualitative network models can aid at understanding how transcriptional regulators and hormonal signaling pathways are dynamically coupled and may underlie both the acquisition of cell fate and the emergence of hormonal activity profiles that arise during complex organ development.

Structure of the SHR-SCR heterodimer bound to the BIRD/IDD transcriptional factor JKD.

The plant-specific GAI, RGA and SCR (GRAS) family proteins play critical roles in plant development and signalling. Two GRAS proteins, SHORT-ROOT (SHR) and SCARECROW (SCR), cooperatively direct asymmetric cell division and the patterning of root cell types by transcriptional control in conjunction with BIRD/INDETERMINATE DOMAIN (IDD) transcription factors, although precise details of these specific interactions and actions remain unknown. Here, we present the crystal structures of the SHR-SCR binary and JACKDAW (JKD)/IDD10-SHR-SCR ternary complexes. Each GRAS domain comprises one α/β core subdomain with an α-helical cap that mediates heterodimerization by forming an intermolecular helix bundle. The α/β core subdomain of SHR forms the BIRD binding groove, which specifically recognizes the zinc fingers of JKD. We identified a conserved SHR-binding motif in 13 BIRD/IDD transcription factors. Our results establish a structural basis for GRAS-GRAS and GRAS-BIRD interactions and provide valuable clues towards our understanding of these regulators, which are involved in plant-specific signalling networks.