TRANSPARENT TESTA GLABRA2 defines trichome cell shape by modulating actin cytoskeleton in Arabidopsis thaliana.

The Arabidopsis (Arabidopsis thaliana) TRANSPARENT TESTA GLABRA2 (TTG2) gene encodes a WRKY transcription factor that regulates a range of development events like trichome, seed coat, and atrichoblast formation. Loss-of-function of TTG2 was previously shown to reduce or eliminate trichome specification and branching. Here, we report the identification of an allele of TTG2, ttg2-6. In contrast to the ttg2 mutants described before, ttg2-6 displayed unique trichome phenotypes. Some ttg2-6 mutant trichomes were hyper-branched, whereas others were hypo-branched, distorted, or clustered. Further, we found that in addition to specifically activating R3 MYB transcription factor TRIPTYCHON (TRY) to modulate trichome specification, TTG2 also integrated cytoskeletal signaling to regulate trichome morphogenesis. The ttg2-6 trichomes displayed aberrant cortical microtubules (cMTs) and actin filaments (F-actin) configurations. Moreover, genetic and biochemical analyses showed that TTG2 could directly bind to the promoter and regulate the expression of BRICK1 (BRK1), which encodes a subunit of the actin nucleation promoting complex suppressor of cyclic AMP repressor (SCAR)/Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE). Collectively, taking advantage of ttg2-6, we uncovered a function for TTG2 in facilitating cMTs and F-actin cytoskeleton-dependent trichome development, providing insight into cellular signaling events downstream of the core transcriptional regulation during trichome development in Arabidopsis.

AXR1 modulates trichome morphogenesis through mediating ROP2 stability in Arabidopsis.

Cell differentiation and morphogenesis are crucial for the establishment of diverse cell types and organs in multicellular organisms. Trichome cells offer an excellent paradigm for dissecting the regulatory mechanisms of plant cell differentiation and morphogenesis due to their unique growth characteristics. Here, we report the isolation of an Arabidopsis mutant, aberrantly branched trichome 3-1 (abt3-1), with a reduced trichome branching phenotype. Positional cloning and molecular complementation experiments confirmed that abt3-1 is a new mutant allele of Auxin resistant 1 (AXR1), which encodes the N-terminal half of ubiquitin-activating enzyme E1 and functions in auxin signaling pathway. Meanwhile, we found that transgenic plants expressing constitutively active version of ROP2 (CA-ROP2) caused a reduction of trichome branches, resembling that of abt3-1. ROP2 is a member of Rho GTPase of plants (ROP) family, serving as versatile signaling switches involved in a range of cellular and developmental processes. Our genetic and biochemical analyses showed AXR1 genetically interacted with ROP2 and mediated ROP2 protein stability. The loss of AXR1 aggravated the trichome defects of CA-ROP2 and induced the accumulation of steady-state ROP2. Consistently, elevated AXR1 expression levels suppressed ROP2 expression and partially rescued trichome branching defects in CA-ROP2 plants. Together, our results presented a new mutant allele of AXR1, uncovered the effects of AXR1 and ROP2 during trichome development, and revealed a pathway of ROP2-mediated regulation of plant cell morphogenesis in Arabidopsis.

A conserved gene regulatory network controls root epidermal cell patterning in superrosid species.

In superrosid species, root epidermal cells differentiate into root hair cells and nonhair cells. In some superrosids, the root hair cells and nonhair cells are distributed randomly (Type I pattern), and in others, they are arranged in a position-dependent manner (Type III pattern). The model plant Arabidopsis (Arabidopsis thaliana) adopts the Type III pattern, and the gene regulatory network (GRN) that controls this pattern has been defined. However, it is unclear whether the Type III pattern in other species is controlled by a similar GRN as in Arabidopsis, and it is not known how the different patterns evolved. In this study, we analyzed superrosid species Rhodiola rosea, Boehmeria nivea, and Cucumis sativus for their root epidermal cell patterns. Combining phylogenetics, transcriptomics, and cross-species complementation, we analyzed homologs of the Arabidopsis patterning genes from these species. We identified R. rosea and B. nivea as Type III species and C. sativus as Type I species. We discovered substantial similarities in structure, expression, and function of Arabidopsis patterning gene homologs in R. rosea and B. nivea, and major changes in C. sativus. We propose that in superrosids, diverse Type III species inherited the patterning GRN from a common ancestor, whereas Type I species arose by mutations in multiple lineages.

Identification of new marker genes from plant single-cell RNA-seq data using interpretable machine learning methods.

An essential step in the analysis of single-cell RNA sequencing data is to classify cells into specific cell types using marker genes. In this study, we have developed a machine learning pipeline called single-cell predictive marker (SPmarker) to identify novel cell-type marker genes in the Arabidopsis root. Unlike traditional approaches, our method uses interpretable machine learning models to select marker genes. We have demonstrated that our method can: assign cell types based on cells that were labelled using published methods; project cell types identified by trajectory analysis from one data set to other data sets; and assign cell types based on internal GFP markers. Using SPmarker, we have identified hundreds of new marker genes that were not identified before. As compared to known marker genes, the new marker genes have more orthologous genes identifiable in the corresponding rice single-cell clusters. The new root hair marker genes also include 172 genes with orthologs expressed in root hair cells in five non-Arabidopsis species, which expands the number of marker genes for this cell type by 35-154%. Our results represent a new approach to identifying cell-type marker genes from scRNA-seq data and pave the way for cross-species mapping of scRNA-seq data in plants.

Plant Cell Identity in the Era of Single-Cell Transcriptomics.

High-throughput single-cell transcriptomic approaches have revolutionized our view of gene expression at the level of individual cells, providing new insights into their heterogeneity, identities, and functions. Recently, technical challenges to the application of single-cell transcriptomics to plants have been overcome, and many plant organs and tissues have now been subjected to analyses at single-cell resolution. In this review, we describe these studies and their impact on our understanding of the diversity, differentiation, and activities of plant cells. We particularly highlight their impact on plant cell identity, including unprecedented views of cell transitions and definitions of rare and novel cell types. We also point out current challenges and future opportunities for the application and analyses of single-cell transcriptomics in plants.

Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression in Arabidopsis roots at the single-cell level.

Similar to other complex organisms, plants consist of diverse and specialized cell types. The gain of unique biological functions of these different cell types is the consequence of the establishment of cell-type-specific transcriptional programs. As a necessary step in gaining a deeper understanding of the regulatory mechanisms controlling plant gene expression, we report the use of single-nucleus RNA sequencing (sNucRNA-seq) and single-nucleus assay for transposase accessible chromatin sequencing (sNucATAC-seq) technologies on Arabidopsis roots. The comparison of our single-nucleus transcriptomes to the published protoplast transcriptomes validated the use of nuclei as biological entities to establish plant cell-type-specific transcriptomes. Furthermore, our sNucRNA-seq results uncovered the transcriptomes of additional cell subtypes not identified by single-cell RNA-seq. Similar to our transcriptomic approach, the sNucATAC-seq approach led to the distribution of the Arabidopsis nuclei into distinct clusters, suggesting the differential accessibility of chromatin between groups of cells according to their identity. To reveal the impact of chromatin accessibility on gene expression, we integrated sNucRNA-seq and sNucATAC-seq data and demonstrated that cell-type-specific marker genes display cell-type-specific patterns of chromatin accessibility. Our data suggest that the differential chromatin accessibility is a critical mechanism to regulate gene activity at the cell-type level.

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.

Molecular Basis for a Cell Fate Switch in Response to Impaired Ribosome Biogenesis in the Arabidopsis Root Epidermis.

The Arabidopsis (Arabidopsis thaliana) root epidermis consists of a position-dependent pattern of root hair cells and non-hair cells. Underlying this cell type patterning is a network of transcription factors including a central MYB-basic helix-loop-helix-WD40 complex containing WEREWOLF (WER), GLABRA3 (GL3)/ENHANCER OF GLABRA3, and TRANSPARENT TESTA GLABRA1. In this study, we used a genetic enhancer screen to identify apum23-4, a mutant allele of the ribosome biogenesis factor (RBF) gene ARABIDOPSIS PUMILIO23 (APUM23), which caused prospective root hair cells to instead adopt the non-hair cell fate. We discovered that this cell fate switch relied on MYB23, a MYB protein encoded by a WER target gene and acting redundantly with WER. In the apum23-4 mutant, MYB23 exhibited ectopic expression that was WER independent and instead required ANAC082, a recently identified ribosomal stress response mediator. We examined additional RBF mutants that produced ectopic non-hair cells and determined that this cell fate switch is generally linked to defects in ribosome biogenesis. Furthermore, the flagellin peptide flg22 triggers the ANAC082-MYB23-GL2 pathway. Taken together, our study provides a molecular explanation for root epidermal cell fate switch in response to ribosomal defects and, more generally, it demonstrates a novel regulatory connection between stress conditions and cell fate control in plants.

Novel TTG1 Mutants Modify Root-Hair Pattern Formation in Arabidopsis.

The patterning of root-hair and non-hair epidermal cells in the Arabidopsis root is governed by a network of transcriptional regulators. The central MYB-bHLH-WD40 (MBW) transcriptional complex includes the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). To clarify the role of TTG1, we describe the identification and analysis of two new ttg1 mutants. Each of these mutants contains a single nucleotide change in the TTG1 gene, which causes a single amino-acid substitution in the predicted TTG1 protein and alters root-hair pattern formation. Surprisingly, these new ttg1 mutants exhibit decreased root-hair formation, particularly in the caprice (cpc) mutant background, rather than increased root-hair formation as reported for strong ttg1 mutants. We show that the unique phenotype of these mutants is due to differential effects of the altered TTG1 proteins on target gene expression, associated with a weakened ability to interact with its GLABRA3 bHLH partner. These findings demonstrate the crucial role of TTG1 for the appropriate balance of target gene activation to achieve the proper pattern of epidermal cell types during Arabidopsis root development.

Nitrate regulation of lateral root and root hair development in plants.

Nitrogen (N) is one of the most important macronutrients for plant growth and development. However, the concentration and distribution of N varies in soil due to a variety of environmental factors. In response, higher plants have evolved a developmentally flexible root system to efficiently take up N under N-limited conditions. Over the past decade, significant progress has been made in understanding this form of plant 'root-foraging' behavior, which is controlled by both a local and a long-distance systemic nitrate signaling pathway. In this review, we focus on the key components of nitrate perception, signaling, and transduction and its role in lateral root development. We also highlight recent findings on the molecular mechanisms of the nitrate systemic signaling pathway, including small signaling peptides involved in long-distance shoot-root communication. Furthermore, we summarize the transcription factor networks responsible for nitrate-dependent lateral root and root hair development.

Root Epidermal Cell Patterning Is Modulated by a Critical Residue in the WEREWOLF Transcription Factor.

The Arabidopsis (Arabidopsis thaliana) root epidermis exhibits a position-dependent pattern of root-hair and nonhair cell types. A highly orchestrated network of gene regulatory interactions, including the R2R3-type MYB transcription factor WEREWOLF (WER), is responsible for generating this cell pattern during root development. In this study, we identified a novel wer mutant from a genetic enhancer screen, designated wer-4, that exhibits an abnormal pattern of root-hair and nonhair cells. We established that wer-4 bears a single-residue substitution (D105N) in the DNA-binding R3 MYB repeat of WER, which differentially affects the transcription of WER target genes, including GLABRA2, CAPRICE, TRIPTYCHON, and ENHANCER OF TRY AND CPC1 This modulation of the gene regulatory network leads to altered levels and distributions of cell fate regulators in the differentiating epidermal cells that ultimately generate the abnormal cell-type pattern. We also created several WER variants with substitutions at the Asp-105 position, and these exhibited a variety of gene expression and cell-type pattern alterations, further supporting the critical role of this residue. These findings provide insight into WER protein function and its importance in generating the proper balance of downstream transcriptional factors in the gene regulatory network that establishes root epidermal cell fate.

QUIRKY regulates root epidermal cell patterning through stabilizing SCRAMBLED to control CAPRICE movement in Arabidopsis.

SCM, a leucine-rich repeat receptor-like kinase, is required for root epidermal cells to appropriately interpret their location and generate the proper cell-type pattern during Arabidopsis root development. Here, via a screen for scm-like mutants we describe a new allele of the QKY gene. We find that QKY is required for the appropriate spatial expression of several epidermal cell fate regulators in a similar manner as SCM in roots, and that QKY and SCM are necessary for the efficient movement of CPC between epidermal cells. We also show that turnover of SCM is mediated by a vacuolar degradation pathway triggered by ubiquitination, and that QKY prevents this SCM ubiquitination through their physical interaction. These results suggest that QKY stabilizes SCM through interaction, and this complex facilitates CPC movement between the epidermal cells to help establish the cell-type pattern in the Arabidopsis root epidermis.

Single-Cell RNA Sequencing Resolves Molecular Relationships Among Individual Plant Cells.

Single-cell RNA sequencing (scRNA-seq) has been used extensively to study cell-specific gene expression in animals, but it has not been widely applied to plants. Here, we describe the use of a commercially available droplet-based microfluidics platform for high-throughput scRNA-seq to obtain single-cell transcriptomes from protoplasts of more than 10,000 Arabidopsis (Arabidopsis thaliana) root cells. We find that all major tissues and developmental stages are represented in this single-cell transcriptome population. Further, distinct subpopulations and rare cell types, including putative quiescent center cells, were identified. A focused analysis of root epidermal cell transcriptomes defined developmental trajectories for individual cells progressing from meristematic through mature stages of root-hair and nonhair cell differentiation. In addition, single-cell transcriptomes were obtained from root epidermis mutants, enabling a comparative analysis of gene expression at single-cell resolution and providing an unprecedented view of the impact of the mutated genes. Overall, this study demonstrates the feasibility and utility of scRNA-seq in plants and provides a first-generation gene expression map of the Arabidopsis root at single-cell resolution.

Plant Systems Biology at the Single-Cell Level.

Our understanding of plant biology is increasingly being built upon studies using 'omics and system biology approaches performed at the level of the entire plant, organ, or tissue. Although these approaches open new avenues to better understand plant biology, they suffer from the cellular complexity of the analyzed sample. Recent methodological advances now allow plant scientists to overcome this limitation and enable biological analyses of single-cells or single-cell-types. Coupled with the development of bioinformatics and functional genomics resources, these studies provide opportunities for high-resolution systems analyses of plant phenomena. In this review, we describe the recent advances, current challenges, and future directions in exploring the biology of single-cells and single-cell-types to enhance our understanding of plant biology as a system.

Positioning of the SCRAMBLED receptor requires UDP-Glc:sterol glucosyltransferase 80B1 in Arabidopsis roots.

The biological function of sterol glucosides (SGs), the most abundant sterol derivatives in higher plants, remains uncertain. In an effort to improve our understanding of these membrane lipids we examined phenotypes exhibited by the roots of Arabidopsis (Arabidopsis thaliana) lines carrying insertions in the UDP-Glc:sterol glucosyltransferase genes, UGT80A2 and UGT80B1. We show that although ugt80A2 mutants exhibit significantly lower levels of total SGs they are morphologically indistinguishable from wild-type plants. In contrast, the roots of ugt80B1 mutants are only deficient in stigmasteryl glucosides but exhibit a significant reduction in root hairs. Sub-cellular investigations reveal that the plasma membrane cell fate regulator, SCRAMBLED (SCM), is mislocalized in ugt80B1 mutants, underscoring the aberrant root epidermal cell patterning. Live imaging of roots indicates that SCM:GFP is localized to the cytoplasm in a non cell type dependent manner instead of the hair (H) cell plasma membrane in these mutants. In addition, we provide evidence for the localization of the UGT80B1 enzyme in the plasma membrane. These data lend further support to the notion that deficiencies in specific SGs are sufficient to disrupt normal cell function and point to a possible role for SGs in cargo transport and/or protein targeting to the plasma membrane.

Diversification of Root Hair Development Genes in Vascular Plants.

The molecular genetic program for root hair development has been studied intensively in Arabidopsis (Arabidopsis thaliana). To understand the extent to which this program might operate in other plants, we conducted a large-scale comparative analysis of root hair development genes from diverse vascular plants, including eudicots, monocots, and a lycophyte. Combining phylogenetics and transcriptomics, we discovered conservation of a core set of root hair genes across all vascular plants, which may derive from an ancient program for unidirectional cell growth coopted for root hair development during vascular plant evolution. Interestingly, we also discovered preferential diversification in the structure and expression of root hair development genes, relative to other root hair- and root-expressed genes, among these species. These differences enabled the definition of sets of genes and gene functions that were acquired or lost in specific lineages during vascular plant evolution. In particular, we found substantial divergence in the structure and expression of genes used for root hair patterning, suggesting that the Arabidopsis transcriptional regulatory mechanism is not shared by other species. To our knowledge, this study provides the first comprehensive view of gene expression in a single plant cell type across multiple species.

Multiple phytohormones promote root hair elongation by regulating a similar set of genes in the root epidermis in Arabidopsis.

Multiple phytohormones, including auxin, ethylene, and cytokinin, play vital roles in regulating cell development in the root epidermis. However, their interactions in specific root hair cell developmental stages are largely unexplored. To bridge this gap, we employed genetic and pharmacological approaches as well as transcriptional analysis in order to dissect their distinct and overlapping roles in root hair initiation and elongation in Arabidopsis thaliana Our results show that among auxin, ethylene, and cytokinin, only ethylene induces ectopic root hair cells in wild-type plants, implying a special role of ethylene in the hair initiation stage. In the subsequent elongation stage, however, auxin, ethylene, and cytokinin enhance root hair tip growth equally. Our data also suggest that the effect of cytokinin is independent from auxin and ethylene in this process. Exogenous cytokinin restores root hair elongation when the auxin and ethylene signal is defective, whereas auxin and ethylene also sustain elongation in the absence of the cytokinin signal. Notably, transcriptional analyses demonstrated that auxin, ethylene, and cytokinin regulate a similar set of root hair-specific genes. Together these analyses provide important clues regarding the mechanism of hormonal interactions and regulation in the formation of single-cell structures.

A single amino acid substitution in the R3 domain of GLABRA1 leads to inhibition of trichome formation in Arabidopsis without affecting its interaction with GLABRA3.

GLABRA1 (GL1) is an R2R3 MYB transcription factor that regulates trichome formation in Arabidopsis by interacting with the bHLH transcription factor GLABRA3 (GL3) or ENHANCER OF GL3 (EGL3). The conserved [D/E]L×2 [R/K]×3L×6L×3R amino acid signature in the R3 domain of MYB proteins has been shown to be required for the interaction of MYBs with R/B-like bHLH transcription factors. By using genetic and molecular analyses, we show that the glabrous phenotype in the nph4-1 mutant is caused by a single nucleotide mutation in the GL1 gene, generating a Ser to Phe substitution (S92F) in the conserved [D/E]L×2[R/K]×3L×6L×3R amino acid signature of GL1. Activation of the integrated GL2p:GUS reporter gene in protoplasts by cotransfection of GL1 and GL3 or EGL3 was abolished by this GL1-S92F substitution. However, GL1-S92F interacted successfully with GL3 or EGL3 in protoplast transfection assays. Unlike VPGL1GL3, the fusion protein VPGL1-S92FGL3 failed to activate the integrated GL2p:GUS reporter gene in transfected protoplasts. These results suggested that the S92 in the conserved [D/E]L×2 [R/K]×3L×6L×3R amino acid signature of GL1 is not essential for the interaction of GL1 and GL3, but may play a role in the binding of GL1 to the promoters of its target genes.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.