RESUMEN
Signal transduction relies largely on the activity of kinases and phosphatases that control protein phosphorylation. However, we still know very little about phosphorylation-mediated signaling networks. Plant MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASEs (MAP4Ks) have recently gained more attention, given their role in a wide range of processes, including developmental processes and stress signaling. We analyzed MAP4K expression patterns and mapped protein-MAP4K interactions in Arabidopsis (Arabidopsis thaliana), revealing extensive coexpression and heterodimerization. This heterodimerization is regulated by the C-terminal, intrinsically disordered half of the MAP4K, and specifically by the coiled coil motif. The ability to heterodimerize is required for proper activity and localization of the MAP4Ks. Taken together, our results identify MAP4K-interacting proteins and emphasize the functional importance of MAP4K heterodimerization. Furthermore, we identified MAP4K4/TARGET OF TEMPERATURE3 (TOT3) and MAP4K5/TOT3-INTERACTING PROTEIN 5 (TOI5) as key regulators of the transition from cell division to elongation zones in the primary root tip.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Multimerización de Proteína , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Dominios Proteicos , Fosforilación , Plantas Modificadas GenéticamenteRESUMEN
SignificanceChemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.
Asunto(s)
Brasinoesteroides/metabolismo , Ácidos Indolacéticos/metabolismo , Proteoma , Transducción de Señal , Aminopiridinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Estabilidad Proteica , Proteómica/métodos , Succinatos/metabolismoRESUMEN
Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Proteínas de Arabidopsis/metabolismo , Temperatura , Proteínas F-Box/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The anaphase promoting complex/cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CELL DIVISION PROTEIN20 (CDC20) and CELL CYCLE SWITCH52 (CCS52). Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis (Arabidopsis thaliana) CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhance the scored ccs52a2-1 phenotypes. Furthermore, whereas the CYCA3;4 protein is promptly broken down after prophase in wild-type plants, it remains present in later stages of mitosis in ccs52a2-1 mutant plants, marking it as a putative APC/CCCS52A2 substrate. Strikingly, increased CYCA3;4 levels result in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4 gain-of-function plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 contributes to disorganized formative divisions, possibly in part caused by the inactivation of RBR1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Proteínas de Ciclo Celular/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , División Celular , Metanosulfonato de Etilo/farmacología , Regulación de la Expresión Génica de las Plantas , Meristema/citología , Meristema/genética , Mutación , Fosforilación , Células Vegetales/efectos de los fármacos , Hojas de la Planta/citología , Hojas de la Planta/genética , Raíces de Plantas/citología , Raíces de Plantas/genética , Tallos de la Planta/citología , Plantas Modificadas Genéticamente , Polimorfismo de Nucleótido SimpleRESUMEN
Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.
Asunto(s)
Adaptación Fisiológica , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Ácidos Indolacéticos/metabolismo , Péptidos/metabolismo , Proteómica , Estrés Fisiológico , Adaptación Fisiológica/genética , Arabidopsis/genética , Transporte Biológico/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Ósmosis , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Plantones/crecimiento & desarrollo , Estrés Fisiológico/genética , Transcripción GenéticaRESUMEN
In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , División Celular , Raíces de Plantas , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , División Celular/genética , División Celular/fisiología , Pared Celular/genética , Pared Celular/fisiología , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , TranscriptomaRESUMEN
In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/citología , División Celular/fisiología , Fosfoproteínas Fosfatasas/fisiología , Raíces de Plantas/citología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Superficie Celular/fisiología , Diferenciación Celular , FosforilaciónRESUMEN
Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity.
Asunto(s)
Calor/efectos adversos , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Triticum/metabolismo , Flores/metabolismo , Hojas de la Planta/metabolismo , Estrés FisiológicoRESUMEN
Leaf growth is a complex, quantitative trait, controlled by a plethora of regulatory mechanisms. Diverse environmental stimuli inhibit leaf growth to cope with the perceived stress. In plant research, mannitol is often used to impose osmotic stress and study the underlying growth-repressing mechanisms. In growing leaf tissue of plants briefly exposed to mannitol-induced stress, a highly interconnected gene regulatory network is induced. However, early signalling and associated protein phosphorylation events that probably precede part of these transcriptional changes and that potentially act at the onset of mannitol-induced leaf size reduction are largely unknown. Here, we performed a proteome and phosphoproteome analysis on growing leaf tissue of Arabidopsis thaliana plants exposed to mild mannitol-induced stress and captured the fast (within the first half hour) events associated with this stress. Based on this in-depth data analysis, 167 and 172 differentially regulated proteins and phosphorylated sites were found. We provide these data sets as a community resource and we flag differentially phosphorylated proteins with described growth-regulatory functions, but we also illustrate potential novel regulators of shoot growth.
Asunto(s)
Arabidopsis/efectos de los fármacos , Manitol/farmacología , Fosfoproteínas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/efectos de los fármacos , Proteoma/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Hojas de la Planta/metabolismo , Proteoma/metabolismoRESUMEN
Plants have developed a variety of mechanisms to cope with abiotic and biotic stresses. In a previous subcellular localization study of hydrogen peroxide-responsive proteins, two peptides with an unknown function (designated ARACIN1 and ARACIN2) have been identified. These peptides are structurally very similar but are transcriptionally differentially regulated during abiotic stresses during Botrytis cinerea infection or after benzothiadiazole and methyl jasmonate treatments. In Arabidopsis (Arabidopsis thaliana), these paralogous genes are positioned in tandem within a cluster of pathogen defense-related genes. Both ARACINs are small, cationic, and hydrophobic peptides, known characteristics for antimicrobial peptides. Their genes are expressed in peripheral cell layers prone to pathogen entry and are lineage specific to the Brassicaceae family. In vitro bioassays demonstrated that both ARACIN peptides have a direct antifungal effect against the agronomically and economically important necrotrophic fungi B. cinerea, Alternaria brassicicola, Fusarium graminearum, and Sclerotinia sclerotiorum and yeast (Saccharomyces cerevisiae). In addition, transgenic Arabidopsis plants that ectopically express ARACIN1 are protected better against infections with both B. cinerea and A. brassicicola. Therefore, we can conclude that both ARACINs act as antimicrobial peptides.
Asunto(s)
Antifúngicos/farmacología , Arabidopsis/microbiología , Brassicaceae/metabolismo , Péptidos/farmacología , Alternaria/efectos de los fármacos , Alternaria/crecimiento & desarrollo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Secuencia de Bases , Botrytis/efectos de los fármacos , Botrytis/crecimiento & desarrollo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Especificidad de Órganos/efectos de los fármacos , Péptidos/química , Péptidos/genética , Fenotipo , Reguladores del Crecimiento de las Plantas/farmacología , Regiones Promotoras Genéticas/genética , Especificidad de la Especie , Estrés Fisiológico/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hormonas Peptídicas/genética , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , División Celular , Hormonas Peptídicas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Roots explore the soil for water and nutrients through the continuous production of lateral roots. Lateral roots are formed at regular distances in a steadily elongating organ, but how future sites for lateral root formation become established is not yet understood. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots. In addition, based on genetic and expression data, we found evidence for the involvement of its proposed receptor, XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1), during the process of lateral root initiation. In conclusion, we report here on the existence of a peptide ligand-receptor kinase interaction that impacts lateral root initiation. Our results represent an important step towards the understanding of the cellular communication implicated in the early phases of lateral root formation.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/crecimiento & desarrollo , Receptores de Péptidos/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
Metacaspases are distant relatives of the metazoan caspases, found in plants, fungi, and protists. However, in contrast with caspases, information about the physiological substrates of metacaspases is still scarce. By means of N-terminal combined fractional diagonal chromatography, the physiological substrates of metacaspase9 (MC9; AT5G04200) were identified in young seedlings of Arabidopsis thaliana on the proteome-wide level, providing additional insight into MC9 cleavage specificity and revealing a previously unknown preference for acidic residues at the substrate prime site position P1'. The functionalities of the identified MC9 substrates hinted at metacaspase functions other than those related to cell death. These results allowed us to resolve the substrate specificity of MC9 in more detail and indicated that the activity of phosphoenolpyruvate carboxykinase 1 (AT4G37870), a key enzyme in gluconeogenesis, is enhanced upon MC9-dependent proteolysis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Caspasas/metabolismo , Proteolisis , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biocatálisis , Caspasas/genética , Regulación de la Expresión Génica de las Plantas , Gluconeogénesis , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Péptidos/química , Péptidos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Plantas Modificadas Genéticamente , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Fracciones Subcelulares/enzimología , Especificidad por SustratoRESUMEN
Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain-containing no apical meristem/Arabidopsis transcription activation factor/cup-shaped cotyledon transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Sitios de Unión , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Perfilación de la Expresión Génica , Mitocondrias/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Paraquat/farmacología , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Unión Proteica , Rotenona/farmacología , Plantones/efectos de los fármacos , Plantones/genética , Plantones/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Plant roots are important for a wide range of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface with the soil environment. Several small signalling peptides and receptor kinases have been shown to affect primary root growth, but very little is known about their role in lateral root development. In this context, the CLE family, a group of small signalling peptides that has been shown to affect a wide range of developmental processes, were the focus of this study. Here, the expression pattern during lateral root initiation for several CLE family members is explored and to what extent CLE1, CLE4, CLE7, CLE26, and CLE27, which show specific expression patterns in the root, are involved in regulating root architecture in Arabidopsis thaliana is assessed. Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling. In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed. Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brachypodium/genética , Regulación de la Expresión Génica de las Plantas , Triticum/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Brachypodium/metabolismo , Filogenia , Raíces de Plantas/metabolismo , Triticum/metabolismoRESUMEN
The development of a multicellular embryo from a single zygote is a complex and highly organized process that is far from understood. In higher plants, apical-basal patterning mechanisms are crucial to correctly specify root and shoot stem cell niches that will sustain and drive post-embryonic plant growth and development. The auxin-responsive AtWRKY23 transcription factor is expressed from early embryogenesis onwards and the timing and localization of its expression overlaps with the root stem cell niche. Knocking down WRKY23 transcript levels or expression of a dominant-negative WRKY23 version via a translational fusion with the SRDX repressor domain affected both apical-basal axis formation as well as installation of the root stem cell niche. WRKY23 expression is affected by two well-known root stem cell specification mechanisms, that is, SHORTROOT and MONOPTEROS-BODENLOS signalling and can partially rescue the root-forming inability of mp embryos. On the basis of these results, we postulate that a tightly controlled WRKY23 expression is involved in the regulation of both auxin-dependent and auxin-independent signalling pathways towards stem cell specification.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción/metabolismo , Arabidopsis/embriología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Meristema/embriología , Meristema/metabolismo , Nicho de Células Madre , Factores de Transcripción/genéticaRESUMEN
Environmental stresses adversely affect plant growth and development. A common theme within these adverse conditions is the perturbation of reactive oxygen species (ROS) homeostasis. Here, we demonstrate that the ROS-inducible Arabidopsis thaliana WRKY15 transcription factor (AtWRKY15) modulates plant growth and salt/osmotic stress responses. By transcriptome profiling, a divergent stress response was identified in transgenic WRKY15-overexpressing plants that linked a stimulated endoplasmic reticulum-to-nucleus communication to a disrupted mitochondrial stress response under salt-stress conditions. We show that mitochondrial calcium-flux sensing might be important for regulating an active mitochondrial retrograde signaling and launching an appropriate defense response to confer salt-stress tolerance.
Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Mitocondrias/metabolismo , Estrés Fisiológico/fisiología , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Calcio/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Análisis por Micromatrices , Mitocondrias/fisiología , Mutagénesis Sitio-Dirigida , Presión Osmótica , Reacción en Cadena en Tiempo Real de la Polimerasa , Salinidad , Factores de Transcripción/genéticaRESUMEN
Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H(2)O(2)-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.
Asunto(s)
Arabidopsis/genética , Daño del ADN , ADN de Plantas/genética , Especies Reactivas de Oxígeno/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidasas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas de las Plantas/genética , Análisis por Conglomerados , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno/farmacología , Immunoblotting , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Peroxidasas/genética , Peroxidasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Reactive oxygen species and redox signaling undergo synergistic and antagonistic interactions with phytohormones to regulate protective responses of plants against biotic and abiotic stresses. However, molecular insight into the nature of this crosstalk remains scarce. We demonstrate that the hydrogen peroxide-responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). Biochemical characterization of recombinant UGT74E2 demonstrated that it strongly favors IBA as a substrate. Assessment of indole-3-acetic acid (IAA), IBA, and their conjugates in transgenic plants ectopically expressing UGT74E2 indicated that the catalytic specificity was maintained in planta. In these transgenic plants, not only were IBA-Glc concentrations increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This perturbed IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Glucosiltransferasas/metabolismo , Indoles/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clonación Molecular , Deshidratación , Glucosiltransferasas/genética , Homeostasis , Ácidos Indolacéticos/metabolismo , Mutagénesis Insercional , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Estrés FisiológicoRESUMEN
Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases.