RESUMEN
Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the Arabidopsis thaliana TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF OBP3-RESPONSIVE GENE 3 (ORG3). While grf9 knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing GRF9 (GRF9ox). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that GRF9 restricts cell proliferation in the early developing leaf. Performing in vitro binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified ORG3 as a direct downstream, and positively regulated target of GRF9. Genetic analysis of grf9 org3 and GRF9ox org3 double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Hojas de la Planta/crecimiento & desarrollo , Proteínas 14-3-3/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión/genética , Proliferación Celular/genética , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes/fisiología , Hojas de la Planta/citología , Plantas Modificadas Genéticamente , Unión Proteica/genéticaRESUMEN
Abiotic stresses cause oxidative damage in plants. Here, we demonstrate that foliar application of an extract from the seaweed Ascophyllum nodosum, SuperFifty (SF), largely prevents paraquat (PQ)-induced oxidative stress in Arabidopsis thaliana. While PQ-stressed plants develop necrotic lesions, plants pre-treated with SF (i.e., primed plants) were unaffected by PQ. Transcriptome analysis revealed induction of reactive oxygen species (ROS) marker genes, genes involved in ROS-induced programmed cell death, and autophagy-related genes after PQ treatment. These changes did not occur in PQ-stressed plants primed with SF. In contrast, upregulation of several carbohydrate metabolism genes, growth, and hormone signaling as well as antioxidant-related genes were specific to SF-primed plants. Metabolomic analyses revealed accumulation of the stress-protective metabolite maltose and the tricarboxylic acid cycle intermediates fumarate and malate in SF-primed plants. Lipidome analysis indicated that those lipids associated with oxidative stress-induced cell death and chloroplast degradation, such as triacylglycerols (TAGs), declined upon SF priming. Our study demonstrated that SF confers tolerance to PQ-induced oxidative stress in A. thaliana, an effect achieved by modulating a range of processes at the transcriptomic, metabolic, and lipid levels.
Asunto(s)
Antioxidantes/farmacología , Arabidopsis/efectos de los fármacos , Ascophyllum/química , Estrés Oxidativo , Extractos Vegetales/farmacología , Transcriptoma , Arabidopsis/genética , Arabidopsis/metabolismo , Metabolismo de los Hidratos de Carbono , Regulación de la Expresión Génica de las Plantas , Herbicidas/toxicidad , Metabolismo de los Lípidos , Paraquat/toxicidadRESUMEN
The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.
Asunto(s)
Arabidopsis/genética , ADN de Plantas/aislamiento & purificación , Formaldehído/farmacología , Biología Molecular/métodos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Arabidopsis/efectos de los fármacos , Cromosomas de las Plantas/genética , ADN de Plantas/genética , Genes Esenciales , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Reacción en Cadena de la Polimerasa , SonicaciónRESUMEN
The growth of plant organs is driven by cell division and subsequent cell expansion. The transition from proliferation to expansion is critical for the final organ size and plant yield. Exit from proliferation and onset of expansion is accompanied by major metabolic reprogramming, and in leaves with the establishment of photosynthesis. To learn more about the molecular mechanisms underlying the developmental and metabolic transitions important for plant growth, we used untargeted proteomics and metabolomics analyses to profile young leaves of a model plant Arabidopsis thaliana representing proliferation, transition, and expansion stages. The dataset presented represents a unique resource comprising approximately 4,000 proteins and 300 annotated small-molecular compounds measured across 6 consecutive days of leaf growth. These can now be mined for novel developmental and metabolic regulators of plant growth and can act as a blueprint for studies aimed at better defining the interface of development and metabolism in other species.
RESUMEN
Growth-regulating factors (GRFs) are plant-specific transcription factors that were originally identified for their roles in stem and leaf development, but recent studies highlight them to be similarly important for other central developmental processes including flower and seed formation, root development, and the coordination of growth processes under adverse environmental conditions. The expression of several GRFs is controlled by microRNA miR396, and the GRF-miRNA396 regulatory module appears to be central to several of these processes. In addition, transcription factors upstream of GRFs and miR396 have been discovered, and gradually downstream target genes of GRFs are being unraveled. Here, we review the current knowledge of the biological functions performed by GRFs and survey available molecular data to illustrate how they exert their roles at the cellular level.
Asunto(s)
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Factores de Transcripción/metabolismo , Flores/crecimiento & desarrollo , Desarrollo de la Planta , Proteínas de Plantas/química , Plantas/genética , Factores de Transcripción/químicaRESUMEN
Salinity negatively affects plant growth and disturbs chloroplast integrity. Here, we aimed at identifying salt-responsive translation-related genes in Arabidopsis thaliana with an emphasis on those encoding plastid-located proteins. We used quantitative real-time PCR to test the expression of 170 genes after short-term salt stress (up to 24 h) and identified several genes affected by the stress including: PRPL11, encoding plastid ribosomal protein L11, ATAB2, encoding a chloroplast-located RNA-binding protein presumably functioning as an activator of translation, and PDF1B, encoding a peptide deformylase involved in N-formyl group removal from nascent proteins synthesized in chloroplasts. These genes were previously shown to have important functions in chloroplast biology and may therefore represent new targets for biotechnological optimization of salinity tolerance.