RESUMEN
The use of single cell sequencing technologies has exploded over recent years, and is now commonly used in many non-model species. Sequencing nuclei instead of whole cells has become increasingly popular, as it does not require the processing of samples immediately after collection. Here we present a highly effective nucleus isolation protocol that outperforms previously available method in challenging samples in a non-model specie. This protocol can be successfully applied to extract nuclei from a variety of tissues and species.
Asunto(s)
Salmo salar , Animales , Núcleo Celular/genética , TecnologíaRESUMEN
Plants are plastic organisms that optimize growth in response to a changing environment. This adaptive capability is regulated by external cues, including light, which provides vital information about the habitat. Phytochrome photoreceptors detect far-red light, indicative of nearby vegetation, and elicit the adaptive shade-avoidance syndrome (SAS), which is critical for plant survival. Plants exhibiting SAS are typically more elongated, with distinctive, small, narrow leaf blades. By applying SAS-inducing end-of-day far-red (EoD FR) treatments at different times during Arabidopsis (Arabidopsis thaliana) leaf 3 development, we have shown that SAS restricts leaf blade size through two distinct cellular strategies. Early SAS induction limits cell division, while later exposure limits cell expansion. This flexible strategy enables phytochromes to maintain control of leaf size through the proliferative and expansion phases of leaf growth. mRNAseq time course data, accessible through a community resource, coupled to a bioinformatics pipeline, identified pathways that underlie these dramatic changes in leaf growth. Phytochrome regulates a suite of major development pathways that control cell division, expansion, and cell fate. Further, phytochromes control cell proliferation through synchronous regulation of the cell cycle, DNA replication, DNA repair, and cytokinesis, and play an important role in sustaining ribosome biogenesis and translation throughout leaf development.
Asunto(s)
Proteínas de Arabidopsis/efectos de la radiación , Arabidopsis/fisiología , Fitocromo/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/metabolismo , División Celular , Luz , Fotorreceptores de Plantas/metabolismo , Fotorreceptores de Plantas/efectos de la radiación , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiaciónRESUMEN
Phytochrome photoreceptors are known to regulate plastic growth responses to vegetation shade. However, recent reports also suggest an important role for phytochromes in carbon resource management, metabolism, and growth. Here, we use 13CO2 labelling patterns in multiallele phy mutants to investigate the role of phytochrome in the control of metabolic fluxes. We also combine quantitative data of 13C incorporation into protein and cell wall polymers, gas exchange measurements, and system modelling to investigate why biomass is decreased in adult multiallele phy mutants. Phytochrome influences the synthesis of stress metabolites such as raffinose and proline, and the accumulation of sugars, possibly through regulating vacuolar sugar transport. Remarkably, despite their modified metabolism and vastly altered architecture, growth rates in adult phy mutants resemble those of wild-type plants. Our results point to delayed seedling growth and smaller cotyledon size as the cause of the adult-stage phy mutant biomass defect. Our data signify a role for phytochrome in metabolic stress physiology and carbon partitioning, and illustrate that phytochrome action at the seedling stage sets the trajectory for adult biomass production.
Asunto(s)
Fitocromo , Plantones/crecimiento & desarrollo , Biomasa , Cotiledón , Luz , Fitocromo B , Estrés FisiológicoRESUMEN
Activation of systemic acquired resistance in plants is associated with transcriptome reprogramming induced by the unstable coactivator NPR1. Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target promoters, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. Here we show ubiquitination of NPR1 is a progressive event in which initial modification by a Cullin-RING E3 ligase promotes its chromatin association and expression of target genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 longevity. Thus, immune-induced transcriptome reprogramming requires sequential actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune activity of NPR1 without strict requirement for its sacrificial turnover.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Productos Agrícolas/inmunología , Transcripción Genética , Ubiquitinación , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.
Asunto(s)
Inmunidad de la Planta/inmunología , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ligasas/genética , Ligasas/metabolismo , Inmunidad de la Planta/fisiología , Poliubiquitina/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ácido Salicílico/metabolismo , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , UbiquitinaciónRESUMEN
Plant immune responses against biotrophic pathogens are regulated by the signaling hormone salicylic acid (SA). SA establishes immunity by regulating a variety of cellular processes, including programmed cell death (PCD) to isolate and kill invading pathogens, and development of systemic acquired resistance (SAR) which provides long-lasting, broad-spectrum resistance throughout the plant. Central to these processes is post-translational modification of SA-regulated signaling proteins by ubiquitination, i.e., the covalent addition of small ubiquitin proteins. Emerging evidence indicates SA-induced protein ubiquitination is largely orchestrated by Cullin-RING ligases (CRLs), which recruit specific substrates for ubiquitination using interchangeable adaptors. Ligation of ubiquitin chains interlinked at lysine 48 leads to substrate degradation by the 26S proteasome. Here we discuss how CRL-mediated degradation of both nucleotide-binding/leucine-rich repeat domain containing immune receptors and SA-induced transcription regulators are critical for functional PCD and SAR responses, respectively. By placing these recent findings in context of knowledge gained in other eukaryotic model species, we highlight potential alternative roles for processive ubiquitination in regulating the activity of SA-mediated immune responses.