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1.
Annu Rev Genet ; 55: 479-496, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34530637

RESUMO

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.


Assuntos
Células Vegetais , Transcriptoma , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Plantas/genética , Análise de Célula Única , Transcriptoma/genética
2.
Plant Cell ; 32(7): 2402-2423, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32371546

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Epiderme Vegetal/citologia , Raízes de Plantas/citologia , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mutação , Epiderme Vegetal/fisiologia , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Ribossomos/metabolismo , Fatores de Transcrição/genética
3.
Plant Cell ; 32(5): 1519-1535, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32111671

RESUMO

Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning process. In the Arabidopsis (Arabidopsis thaliana) root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells (CCs) and the other for proto- and metaphloem sieve elements (SEs). The molecular mechanisms underlying each of these processes have been reported; however, how these are coordinated has remained elusive. Here, we report a new phloem development process coordinated via the SHORTROOT (SHR) transcription factor in Arabidopsis. The movement of SHR into the endodermis regulates the ACD for CC formation by activating microRNA165/6, while SHR moving into the phloem regulates the ACD generating the two phloem SEs. In the phloem, SHR sequentially activates NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 2 (SND2), and these three together form a positive feedforward loop. Under this regulatory scheme, NARS1, generated in the CCs of the root differentiation zone, establishes a top-down signal that drives the ACD for phloem SEs in the meristem. SND2 appears to function downstream to amplify NARS1 via positive feedback. This new regulatory mechanism expands our understanding of the sophisticated vascular tissue patterning processes occurring during postembryonic root development.plantcell;32/5/1519/FX1F1fx1.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Floema/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Divisão Celular Assimétrica , Diferenciação Celular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , MicroRNAs/genética , MicroRNAs/metabolismo , Floema/citologia , Floema/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Fatores de Transcrição/genética
4.
Plant Physiol ; 181(3): 1239-1256, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31492737

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo
5.
Plant Physiol ; 179(4): 1444-1456, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718350

RESUMO

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.


Assuntos
Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Análise de Célula Única , Transcriptoma , Arabidopsis/citologia , Estudos de Viabilidade , Epiderme Vegetal/metabolismo , Raízes de Plantas/citologia , Protoplastos/metabolismo , Análise de Sequência de RNA
6.
Plant Physiol ; 174(3): 1697-1712, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28487476

RESUMO

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.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Genes Controladores do Desenvolvimento , Genes de Plantas , Variação Genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/metabolismo , Funções Verossimilhança , Morfogênese/genética , Família Multigênica , Oryza/genética , Filogenia , Especificidade da Espécie
7.
PLoS Genet ; 11(3): e1004973, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25730098

RESUMO

Plant growth depends on stem cell niches in meristems. In the root apical meristem, the quiescent center (QC) cells form a niche together with the surrounding stem cells. Stem cells produce daughter cells that are displaced into a transit-amplifying (TA) domain of the root meristem. TA cells divide several times to provide cells for growth. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of the stem cell niche. Cytokinin controls TA cell activities in a dose-dependent manner. Although the regulatory programs in each compartment of the root meristem have been identified, it is still unclear how they coordinate one another. Here, we investigate how PHABULOSA (PHB), under the posttranscriptional control of SHR and SCR, regulates TA cell activities. The root meristem and growth defects in shr or scr mutants were significantly recovered in the shr phb or scr phb double mutant, respectively. This rescue in root growth occurs in the absence of a QC. Conversely, when the modified PHB, which is highly resistant to microRNA, was expressed throughout the stele of the wild-type root meristem, root growth became very similar to that observed in the shr; however, the identity of the QC was unaffected. Interestingly, a moderate increase in PHB resulted in a root meristem phenotype similar to that observed following the application of high levels of cytokinin. Our protoplast assay and transgenic approach using ARR10 suggest that the depletion of TA cells by high PHB in the stele occurs via the repression of B-ARR activities. This regulatory mechanism seems to help to maintain the cytokinin homeostasis in the meristem. Taken together, our study suggests that PHB can dynamically regulate TA cell activities in a QC-independent manner, and that the SHR-PHB pathway enables a robust root growth system by coordinating the stem cell niche and TA domain.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Homeodomínio/genética , Meristema/genética , Nicho de Células-Tronco/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/metabolismo , Divisão Celular/genética , Citocininas/genética , Citocininas/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/biossíntese , Homeostase , Meristema/crescimento & desenvolvimento , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo
8.
Physiol Plant ; 157(2): 221-33, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26968317

RESUMO

Root apical meristem (RAM) drives post-embryonic root growth by constantly supplying cells through mitosis. It is composed of stem cells and their derivatives, the transit-amplifying (TA) cells. Stem cell organization and its maintenance in the RAM are well characterized, however, their relationships with TA cells remain unclear. SHORTROOT (SHR) is critical for root development. It patterns cell types and promotes the post-embryonic root growth. Defective root growth in the shr has been ascribed to the lack of quiescent center (QC), which maintains the surrounding stem cells. However, our recent investigation indicated that SHR maintains TA cells independently of QC by modulating PHABULOSA (PHB) through miRNA165/6. PHB controls TA cell activity by modulating cytokinin levels and type B Arabidopsis Response Regulator activity, in a dosage-dependent manner. To further understand TA cell regulation, we conducted a shr suppressor screen. With an extensive mutagenesis screen followed by genome sequencing of a pooled F2 population, we discovered two suppressor alleles with mutations in HAWAIIAN SKIRT (HWS). HWS, encoding an F-box protein with kelch domain, is expressed, partly depending on SHR, in the root cap and in the pericycle of the differentiation zone. Interestingly, root growth in the shr hws was more active than the wild-type roots for the first 7 days after germination, without recovering QC. Contrary to shr phb, shr hws did not show a recovery of cytokinin signaling. These indicate that HWS affects QC-independent TA cell activities through a pathway distinctive from PHB.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas F-Box/metabolismo , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Citocininas/metabolismo , Proteínas F-Box/genética , Germinação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Meristema/citologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento
9.
Plant Physiol ; 156(4): 1867-77, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21653190

RESUMO

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Padronização Corporal/genética , Proteínas de Ligação a DNA/metabolismo , Flores/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Estabilidade de RNA/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Mutação/genética , Fotoperíodo , Raízes de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica
10.
Plant Physiol ; 157(3): 1196-208, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21914815

RESUMO

The root hair and nonhair cells in the Arabidopsis (Arabidopsis thaliana) root epidermis are specified by a suite of transcriptional regulators. Two of these are WEREWOLF (WER) and CAPRICE (CPC), which encode MYB transcription factors that are required for promoting the nonhair cell fate and the hair cell fate, respectively. However, the precise function and relationship between these transcriptional regulators have not been fully defined experimentally. Here, we examine these issues by misexpressing the WER gene using the GAL4-upstream activation sequence transactivation system. We find that WER overexpression in the Arabidopsis root tip is sufficient to cause epidermal cells to adopt the nonhair cell fate through direct induction of GLABRA2 (GL2) gene expression. We also show that GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), two closely related bHLH proteins, are required for the action of the overexpressed WER and that WER interacts with these bHLHs in plant cells. Furthermore, we find that CPC suppresses the WER overexpression phenotype quantitatively. These results show that WER acts together with GL3/EGL3 to induce GL2 expression and that WER and CPC compete with one another to define cell fates in the Arabidopsis root epidermis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Epiderme Vegetal/citologia , Raízes de Plantas/citologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Células Vegetais/metabolismo , Epiderme Vegetal/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica
11.
Mol Plant ; 14(3): 372-383, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33422696

RESUMO

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.


Assuntos
Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Raízes de Plantas/metabolismo , RNA-Seq , Transcriptoma/genética
12.
Curr Opin Plant Biol ; 16(1): 5-10, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23287386

RESUMO

Cell fate decisions are of primary importance for plant development. Their simple 'either-or' outcome and dynamic nature has attracted the attention of computational modelers. Recent efforts have focused on modeling the determination of several epidermal cell types in the root and shoot of Arabidopsis where many molecular components have been defined. Results of integrated modeling and molecular biology experimentation in these systems have highlighted the importance of competitive positive and negative factors and interconnected feedback loops in generating flexible yet robust mechanisms for establishing distinct gene expression programs in neighboring cells. These models have proven useful in judging hypotheses and guiding future research.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Simulação por Computador , Regulação da Expressão Gênica no Desenvolvimento , Epiderme Vegetal/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Padronização Corporal , Diferenciação Celular , Retroalimentação , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Estômatos de Plantas/citologia , Estômatos de Plantas/genética , Estômatos de Plantas/crescimento & desenvolvimento
13.
Development ; 132(21): 4765-75, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16207757

RESUMO

The Arabidopsis root epidermis is composed of two types of cells, hair cells and non-hair cells, and their fate is determined in a position-dependent manner. WEREWOLF (WER), a R2R3 MYB protein, has been shown genetically to function as a master regulator to control both of the epidermal cell fates. To directly test the proposed role of WER in this system, we examined its subcellular localization and defined its transcriptional activation properties. We show that a WER-GFP fusion protein is functional and accumulates in the nucleus of the N-position cells in the Arabidopsis root epidermis, as expected for a transcriptional regulator. We also find that a modified WER protein with a strong activation domain (WER-VP16) promotes the formation of both epidermal cell types, supporting the view that WER specifies both cell fates. In addition, we used the glucocorticoid receptor (GR) inducible system to show that CPC transcription is regulated directly by WER. Using EMSA, we found two WER-binding sites (WBSs; WBSI and WBSII) in the CPC promoter. WER-WBSI binding was confirmed in vivo using the yeast one-hybrid assay. Binding between the WER protein and both WBSs (WBSI and WBSII), and the importance of the two WBSs in CPC promoter activity were confirmed in Arabidopsis. These results provide experimental support for the proposed role of WER as an activator of gene transcription during the specification of both epidermal cell fates.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/citologia , Linhagem da Célula/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Plantas/genética , Proteínas Proto-Oncogênicas c-myb/genética , Transcrição Gênica , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/citologia , Regiões Promotoras Genéticas
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