RESUMEN
Single-cell proteomics (SCP) is an emerging approach to resolve cellular heterogeneity within complex tissues of multi-cellular organisms. Here, we demonstrate the feasibility of SCP on plant samples using the model plant Arabidopsis thaliana. Specifically, we focused on examining isolated single cells from the cortex and endodermis, which are two adjacent root cell types derived from a common stem cell lineage. From 756 root cells, we identified 3763 proteins and 1118 proteins/cell. Ultimately, we focus on 3217 proteins quantified following stringent filtering. Of these, we identified 596 proteins whose expression is enriched in either the cortex or endodermis and are able to differentiate these closely related plant cell types. Collectivity, this study demonstrates that SCP can resolve neighboring cell types with distinct functions, thereby facilitating the identification of biomarkers and candidate proteins to enable functional genomics.
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Single-cell genomics technologies are ushering in a new research era. In this review, we summarize the benefits and current challenges of using these technologies to probe the transcriptional regulation of plant development. In addition to profiling cells at a single snapshot in time, researchers have recently produced time-resolved datasets to map cell responses to stimuli. Live-imaging and spatial transcriptomic techniques are rapidly being adopted to link a cell's transcriptional profile with its spatial location within a tissue. Combining these technologies is a powerful spatiotemporal approach to investigate cell plasticity and developmental responses that contribute to plant resilience. Although there are hurdles to overcome, we conclude by discussing how single-cell genomics is poised to address developmental questions in the coming years.
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Genómica , Transcriptoma , Genómica/métodos , Perfilación de la Expresión Génica , Desarrollo de la Planta/genética , Análisis de la Célula Individual/métodosRESUMEN
Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.
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Proteínas de Arabidopsis , Brasinoesteroides , Plasmodesmos/metabolismo , Reguladores del Crecimiento de las Plantas , Plantas/metabolismo , Hormonas , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/metabolismoRESUMEN
Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and cell elongation, through gene regulatory networks that vary in space and time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we identified the elongating cortex as a site where brassinosteroids trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 (HAT7) and GT-2-LIKE 1 (GTL1) as brassinosteroid-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as a site of brassinosteroid-mediated growth and unveil a brassinosteroid signaling network regulating the transition from proliferation to elongation, which illuminates aspects of spatiotemporal hormone responses.
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Proteínas de Arabidopsis , Arabidopsis , Brasinoesteroides , Diferenciación Celular , División Celular , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Reguladores del Crecimiento de las Plantas , Raíces de Plantas , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , División Celular/genética , Diferenciación Celular/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
Macroautophagy/autophagy is a conserved recycling process that maintains cellular homeostasis during environmental stress. Autophagy is negatively regulated by TOR (target of rapamycin), a nutrient-regulated protein kinase that in plants is activated by several phytohormones, leading to increased growth. However, the detailed molecular mechanisms by which TOR integrates autophagy and hormone signaling are poorly understood. Here, we show that TOR modulates brassinosteroid (BR)-regulated plant growth and stress-response pathways. Active TOR was required for full BR-mediated growth in Arabidopsis thaliana. Autophagy was constitutively up-regulated upon blocking BR biosynthesis or signaling, and down-regulated by increasing the activity of the BR pathway. BIN2 (brassinosteroid-insensitive 2) kinase, a GSK3-like kinase functioning as a negative regulator in BR signaling, directly phosphorylated RAPTOR1B (regulatory-associated protein of TOR 1B), a substrate-recruiting subunit in the TOR complex, at a conserved serine residue within a typical BIN2 phosphorylation motif. Mutation of RAPTOR1B serine 916 to alanine, to block phosphorylation by BIN2, repressed autophagy and increased phosphorylation of the TOR substrate ATG13a (autophagy-related protein 13a). By contrast, this mutation had only a limited effect on growth. We present a model in which RAPTOR1B is phosphorylated and inhibited by BIN2 when BRs are absent, activating the autophagy pathway. When BRs signal and inhibit BIN2, RAPTOR1B is thus less inhibited by BIN2 phosphorylation. This leads to increased TOR activity and ATG13a phosphorylation, and decreased autophagy activity. Our studies define a new mechanism by which coordination between BR and TOR signaling pathways helps to maintain the balance between plant growth and stress responses.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Fosforilación , Brasinoesteroides/farmacología , Brasinoesteroides/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Arabidopsis/metabolismo , Autofagia , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas/metabolismoRESUMEN
We describe a protocol to perform fast and non-arbitrary quality control of single-cell RNA sequencing (scRNA-seq) raw data using scKB and COPILOT. scKB is a wrapper script of kallisto and bustools for accelerated alignment and transcript count matrix generation, which runs significantly faster than the popular tool Cell Ranger. COPILOT then offers non-arbitrary background noise removal by comparing distributions of low-quality and high-quality cells. Together, this protocol streamlines the processing workflow and provides an easy entry for new scRNA-seq users. For complete details on the use and execution of this protocol, please refer to Shahan et al. (2022).
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Análisis de Expresión Génica de una Sola Célula , Programas Informáticos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Control de CalidadRESUMEN
FERONIA (FER) receptor kinase plays versatile roles in plant growth and development, biotic and abiotic stress responses, and reproduction. Autophagy is a conserved cellular recycling process that is critical for balancing plant growth and stress responses. Target of Rapamycin (TOR) has been shown to be a master regulator of autophagy. Our previous multi-omics analysis with loss-of-function fer-4 mutant implicated that FER functions in the autophagy pathway. We further demonstrated here that the fer-4 mutant displayed constitutive autophagy, and FER is required for TOR kinase activity measured by S6K1 phosphorylation and by root growth inhibition assay to TOR kinase inhibitor AZD8055. Taken together, our study provides a previously unknown mechanism by which FER functions through TOR to negatively regulate autophagy.
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Brassinosteroids (BRs) and Target of Rapamycin Complex (TORC) are two major actors coordinating plant growth and stress responses. Brassinosteroids function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies have revealed connections between these two pathways, but a system-wide view of their interplay is still missing. We quantified the level of 23 975 transcripts, 11 183 proteins, and 27 887 phosphorylation sites in wild-type Arabidopsis thaliana and in mutants with altered levels of either BRASSINOSTEROID INSENSITIVE 2 (BIN2) or REGULATORY ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Furthermore, we used the multi-omic data to reconstruct an integrated signaling network. We screened 41 candidate genes identified from the reconstructed network and found that loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results establish a predictive network that defines different layers of molecular interactions between BR- or TORC-regulated growth and autophagy.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Brasinoesteroides/farmacología , Regulación de la Expresión Génica de las Plantas , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Sirolimus , Factores de Transcripción/metabolismoRESUMEN
The receptor kinase FERONIA (FER) is a versatile regulator of plant growth and development, biotic and abiotic stress responses, and reproduction. To gain new insights into the molecular interplay of these processes and to identify new FER functions, we carried out quantitative transcriptome, proteome, and phosphoproteome profiling of Arabidopsis (Arabidopsis thaliana) wild-type and fer-4 loss-of-function mutant plants. Gene ontology terms for phytohormone signaling, abiotic stress, and biotic stress were significantly enriched among differentially expressed transcripts, differentially abundant proteins, and/or misphosphorylated proteins, in agreement with the known roles for FER in these processes. Analysis of multiomics data and subsequent experimental evidence revealed previously unknown functions for FER in endoplasmic reticulum (ER) body formation and glucosinolate biosynthesis. FER functions through the transcription factor NAI1 to mediate ER body formation. FER also negatively regulates indole glucosinolate biosynthesis, partially through NAI1. Furthermore, we found that a group of abscisic acid (ABA)-induced transcription factors is hypophosphorylated in the fer-4 mutant and demonstrated that FER acts through the transcription factor ABA INSENSITIVE5 (ABI5) to negatively regulate the ABA response during cotyledon greening. Our integrated omics study, therefore, reveals novel functions for FER and provides new insights into the underlying mechanisms of FER function.
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Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Glucosinolatos/metabolismo , Fosfotransferasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In all multicellular organisms, transcriptional networks orchestrate organ development. The Arabidopsis root, with its simple structure and indeterminate growth, is an ideal model for investigating 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.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Redes Reguladoras de Genes/genética , Raíces de Plantas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Redes Reguladoras de Genes/fisiología , Mutación/genética , Raíces de Plantas/metabolismo , Análisis de la Célula Individual/métodos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/fisiologíaRESUMEN
Brassinosteroids (BRs) are plant steroid hormones that regulate cell division and stress response. Here we use a systems biology approach to integrate multi-omic datasets and unravel the molecular signaling events of BR response in Arabidopsis. We profile the levels of 26,669 transcripts, 9,533 protein groups, and 26,617 phosphorylation sites from Arabidopsis seedlings treated with brassinolide (BL) for six different lengths of time. We then construct a network inference pipeline called Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) to integrate these data. We use our network predictions to identify putative phosphorylation sites on BES1 and experimentally validate their importance. Additionally, we identify BRONTOSAURUS (BRON) as a transcription factor that regulates cell division, and we show that BRON expression is modulated by BR-responsive kinases and transcription factors. This work demonstrates the power of integrative network analysis applied to multi-omic data and provides fundamental insights into the molecular signaling events occurring during BR response.
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Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Transducción de Señal , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , División Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteómica , Plantones/metabolismo , Esteroides Heterocíclicos , Factores de Transcripción/metabolismoRESUMEN
Brassinosteroids (BRs) regulate plant growth, development, and stress responses by activating the core transcription factor BRI1-EMS-SUPPRESSOR1 (BES1), whose degradation occurs through the proteasome and autophagy pathways. The E3 ubiquitin ligase(s) that modify BES1 for autophagy-mediated degradation remain to be fully defined. Here, we identified an F-box family E3 ubiquitin ligase named BES1-ASSOCIATED F-BOX1 (BAF1) in Arabidopsis thaliana. BAF1 interacts with BES1 and mediates its ubiquitination and degradation. Our genetic data demonstrated that BAF1 inhibits BR signaling in a BES1-dependent manner. Moreover, BAF1 targets BES1 for autophagic degradation in a selective manner. BAF1-triggered selective autophagy of BES1 depends on the ubiquitin binding receptor DOMINANT SUPPRESSOR OF KAR2 (DSK2). Sucrose starvation-induced selective autophagy of BES1, but not bulk autophagy, was significantly compromised in baf1 mutant and BAF1-ΔF (BAF1 F-box decoy) overexpression plants, but clearly increased by BAF1 overexpression. The baf1 and BAF1-ΔF overexpression plants had increased BR-regulated growth but were sensitive to long-term sucrose starvation, while BAF1 overexpression plants had decreased BR-regulated growth but were highly tolerant of sucrose starvation. Our results not only established BAF1 as an E3 ubiquitin ligase that targets BES1 for degradation through selective autophagy pathway, but also revealed a mechanism for plants to reduce growth during sucrose starvation.
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Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Autofagia , Brasinoesteroides/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligasas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Brassinosteroids (BRs) are a group of plant steroid hormones involved in regulating growth, development, and stress responses. Many components of the BR pathway have previously been identified and characterized. However, BR phenotyping experiments are typically performed in a low-throughput manner, such as on Petri plates. Additionally, the BR pathway affects drought responses, but drought experiments are time consuming and difficult to control. To mitigate these issues and increase throughput, we developed the Robotic Assay for Drought (RoAD) system to perform BR and drought response experiments in soil-grown Arabidopsis plants. RoAD is equipped with a robotic arm, a rover, a bench scale, a precisely controlled watering system, an RGB camera, and a laser profilometer. It performs daily weighing, watering, and imaging tasks and is capable of administering BR response assays by watering plants with Propiconazole (PCZ), a BR biosynthesis inhibitor. We developed image processing algorithms for both plant segmentation and phenotypic trait extraction to accurately measure traits including plant area, plant volume, leaf length, and leaf width. We then applied machine learning algorithms that utilize the extracted phenotypic parameters to identify image-derived traits that can distinguish control, drought-treated, and PCZ-treated plants. We carried out PCZ and drought experiments on a set of BR mutants and Arabidopsis accessions with altered BR responses. Finally, we extended the RoAD assays to perform BR response assays using PCZ in Zea mays (maize) plants. This study establishes an automated and non-invasive robotic imaging system as a tool to accurately measure morphological and growth-related traits of Arabidopsis and maize plants in 3D, providing insights into the BR-mediated control of plant growth and stress responses.
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Arabidopsis/fisiología , Brasinoesteroides/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Robótica/métodos , Zea mays/fisiología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Sequías , Diseño de Equipo , Aprendizaje Automático , Fenotipo , Proteínas Quinasas/genética , Robótica/instrumentación , Plantones/fisiología , Suelo/química , Triazoles/farmacologíaRESUMEN
A fundamental question in developmental biology is how the progeny of stem cells become differentiated tissues. The Arabidopsis root is a tractable model to address this question due to its simple organization and defined cell lineages. In particular, the zone of dividing cells at the root tip-the root apical meristem-presents an opportunity to map the gene regulatory networks underlying stem cell niche maintenance, tissue patterning, and cell identity acquisition. To identify molecular regulators of these processes, studies over the last 20 years employed global profiling of gene expression patterns. However, these technologies are prone to information loss due to averaging gene expression signatures over multiple cell types and/or developmental stages. Recently developed high-throughput methods to profile gene expression at single-cell resolution have been successfully applied to plants. Here, we review insights from the first published single-cell mRNA sequencing and chromatin accessibility datasets generated from Arabidopsis roots. These studies successfully reconstruct developmental trajectories, phenotype cell identity mutants at unprecedented resolution, and reveal cell type-specific responses to environmental stimuli. The experimental insight gained from Arabidopsis paves the way to profile roots from additional species.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Meristema/genética , Raíces de Plantas/genética , Análisis de la Célula IndividualRESUMEN
Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. In Arabidopsis thaliana, the core machinery of autophagy is well defined, but its transcriptional regulation is largely unknown. The ATG8 (autophagy-related 8) protein plays central roles in decorating autophagosomes and binding to specific cargo receptors to recruit cargo to autophagosomes. We propose that the transcriptional control of ATG8 genes is important during the formation of autophagosomes and therefore contributes to survival during stress. Here, we describe a yeast one-hybrid (Y1H) screen for transcription factors (TFs) that regulate ATG8 gene expression in Arabidopsis, using the promoters of 4 ATG8 genes. We identified a total of 225 TFs from 35 families that bind these promoters. The TF-ATG8 promoter interactions revealed a wide array of diverse TF families for different promoters, as well as enrichment for families of TFs that bound to specific fragments. These TFs are not only involved in plant developmental processes but also in the response to environmental stresses. TGA9 (TGACG (TGA) motif-binding protein 9)/AT1G08320 was confirmed as a positive regulator of autophagy. TGA9 overexpression activated autophagy under both control and stress conditions and transcriptionally up-regulated expression of ATG8B, ATG8E and additional ATG genes via binding to their promoters. Our results provide a comprehensive resource of TFs that regulate ATG8 gene expression and lay a foundation for understanding the transcriptional regulation of plant autophagy.Abbreviations: ABRC: Arabidopsis biological resource center; AP2-EREBP: APETALA2/Ethylene-responsive element binding protein; ARF: auxin response factor; ATF4: activating transcription factor 4; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; DAP-seq: DNA affinity purification sequencing; FOXO: forkhead box O; GFP: green fluorescent protein; GO: gene ontologies; HB: homeobox; LD: long-day; LUC: firefly luciferase; MAP1LC3: microtubule associated protein 1 light chain 3; MDC: monodansylcadaverine; 3-MA: 3-methyladenine; OE: overexpressing; PCD: programmed cell death; qPCR: quantitative polymerase chain reaction; REN: renilla luciferase; RT: room temperature; SD: standard deviation; TF: transcription factor; TFEB: transcription factor EB; TGA: TGACG motif; TOR: target of rapamycin; TSS: transcription start site; WT: wild-type; Y1H: yeast one-hybrid.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia/fisiología , Factores de Transcripción/metabolismo , Arabidopsis/genética , Autofagosomas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Proteínas de Plantas , Regiones Promotoras Genéticas/genética , Estrés Fisiológico/fisiologíaRESUMEN
Brassinosteroids (BRs) are a group of polyhydroxylated plant steroid hormones that are crucial for many aspects of a plant's life. BRs were originally characterized for their function in cell elongation, but it is becoming clear that they play major roles in plant growth, development, and responses to several stresses such as extreme temperatures and drought. A BR signaling pathway from cell surface receptors to central transcription factors has been well characterized. Here, we summarize recent progress toward understanding the BR pathway, including BR perception and the molecular mechanisms of BR signaling. Next, we discuss the roles of BRs in development and stress responses. Finally, we show how knowledge of the BR pathway is being applied to manipulate the growth and stress responses of crops. These studies highlight the complex regulation of BR signaling, multiple points of crosstalk between BRs and other hormones or stress responses, and the finely tuned spatiotemporal regulation of BR signaling.
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Brasinoesteroides/metabolismo , Desarrollo de la Planta/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Estrés Fisiológico/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Productos Agrícolas , Proteínas de Unión al ADN/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal/fisiología , Factores de Transcripción/metabolismoRESUMEN
Dynamic environmental changes such as extreme temperature, water scarcity and high salinity affect plant growth, survival, and reproduction. Plants have evolved sophisticated regulatory mechanisms to adapt to these unfavorable conditions, many of which interface with plant hormone signaling pathways. Abiotic stresses alter the production and distribution of phytohormones that in turn mediate stress responses at least in part through hormone- and stress-responsive transcription factors. Among these, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) family transcription factors (AP2/ERFs) have emerged as key regulators of various stress responses, in which they also respond to hormones with improved plant survival during stress conditions. Apart from participation in specific stresses, AP2/ERFs are involved in a wide range of stress tolerance, enabling them to form an interconnected stress regulatory network. Additionally, many AP2/ERFs respond to the plant hormones abscisic acid (ABA) and ethylene (ET) to help activate ABA and ET dependent and independent stress-responsive genes. While some AP2/ERFs are implicated in growth and developmental processes mediated by gibberellins (GAs), cytokinins (CTK), and brassinosteroids (BRs). The involvement of AP2/ERFs in hormone signaling adds the complexity of stress regulatory network. In this review, we summarize recent studies on AP2/ERF transcription factors in hormonal and abiotic stress responses with an emphasis on selected family members in Arabidopsis. In addition, we leverage publically available Arabidopsis gene networks and transcriptome data to investigate AP2/ERF regulatory networks, providing context and important clues about the roles of diverse AP2/ERFs in controlling hormone and stress responses.
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Bacterial pathogens use effectors and phytotoxins to facilitate infection of host plants. Coronatine (COR) is one of the phytotoxins produced in bacterial pathogens, such as Pseudomonas syringae pv. tomato DC3000 (pst DC3000). COR structurally and functionally mimics the active form of the plant hormone jasmonic acid (JA), JA-isoleucine (JA-Ile), and can hijack the host JA-signaling pathway to achieve host disease susceptibility [1]. COR utilizes the transcription factor MYC2, a master regulator of JA signaling, to activate NAC transcription factors, which functions to inhibit accumulation of salicylic acid (SA) and thus compromise host immunity [2]. It has been demonstrated that SA can antagonize JA signaling through NONEXPRESSOR of PATHOGENESIS-RELATED GENE1 (NPR1) [3] and downstream transcription factors TGAs [4] and WRKYs [5, 6]. However, the detailed mechanism by which host plants counteract COR-mediated susceptibility is largely unknown. Here, we show that the receptor kinase FERONIA (FER) functions to inhibit JA and COR signaling by phosphorylating and destabilizing MYC2, thereby positively regulating immunity. Conversely, the peptide ligand RALF23 acts through FER to stabilize MYC2 and elevate JA signaling, negatively contributing to plant immunity. Our results establish the RALF23-FER-MYC2 signaling module and provide a previously unknown mechanism by which host plants utilize FER signaling to counteract COR-mediated host disease susceptibility.
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Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Ciclopentanos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Oxilipinas/metabolismo , Fosfotransferasas/genética , Inmunidad de la Planta/genética , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosfotransferasas/metabolismo , Transducción de SeñalRESUMEN
Plant steroid hormones, brassinosteroids (BRs), play important roles in growth and development. BR signaling controls the activities of BRASSINOSTERIOD INSENSITIVE1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 (BES1/BZR1) family transcription factors. Besides the role in promoting growth, BRs are also implicated in plant responses to drought stress. However, the molecular mechanisms by which BRs regulate drought response have just begun to be revealed. The functions of WRKY transcription factors in BR-regulated plant growth have not been established, although their roles in stress responses are well documented. Here, we found that three Arabidopsis thaliana group III WRKY transcription factors, WRKY46, WRKY54, and WRKY70, are involved in both BR-regulated plant growth and drought response as the wrky46 wrky54 wrky70 triple mutant has defects in BR-regulated growth and is more tolerant to drought stress. RNA-sequencing analysis revealed global roles of WRKY46, WRKY54, and WRKY70 in promoting BR-mediated gene expression and inhibiting drought responsive genes. WRKY54 directly interacts with BES1 to cooperatively regulate the expression of target genes. In addition, WRKY54 is phosphorylated and destabilized by GSK3-like kinase BR-INSENSITIVE2, a negative regulator in the BR pathway. Our results therefore establish WRKY46/54/70 as important signaling components that are positively involved in BR-regulated growth and negatively involved in drought responses.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Sequías , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genéticaRESUMEN
Plants encounter a variety of stresses and must fine-tune their growth and stress-response programs to best suit their environment. BES1 functions as a master regulator in the brassinosteroid (BR) pathway that promotes plant growth. Here, we show that BES1 interacts with the ubiquitin receptor protein DSK2 and is targeted to the autophagy pathway during stress via the interaction of DSK2 with ATG8, a ubiquitin-like protein directing autophagosome formation and cargo recruitment. Additionally, DSK2 is phosphorylated by the GSK3-like kinase BIN2, a negative regulator in the BR pathway. BIN2 phosphorylation of DSK2 flanking its ATG8 interacting motifs (AIMs) promotes DSK2-ATG8 interaction, thereby targeting BES1 for degradation. Accordingly, loss-of-function dsk2 mutants accumulate BES1, have altered global gene expression profiles, and have compromised stress responses. Our results thus reveal that plants coordinate growth and stress responses by integrating BR and autophagy pathways and identify the molecular basis of this crosstalk.