RESUMEN
Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.
Asunto(s)
Arabidopsis , Chlamydomonas reinhardtii , Presión Osmótica , Transducción de Señal , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Proteómica , Regulación de la Expresión Génica de las Plantas , Genómica , Estrés Fisiológico , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Transcriptoma , Compartimento Celular , Cloroplastos/metabolismo , MultiómicaRESUMEN
The shape of a plant's root system influences its ability to reach essential nutrients in the soil and to acquire water during drought. Progress in engineering plant roots to optimize water and nutrient acquisition has been limited by our capacity to design and build genetic programs that alter root growth in a predictable manner. We developed a collection of synthetic transcriptional regulators for plants that can be compiled to create genetic circuits. These circuits control gene expression by performing Boolean logic operations and can be used to predictably alter root structure. This work demonstrates the potential of synthetic genetic circuits to control gene expression across tissues and reprogram plant growth.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes Sintéticos , Raíces de Plantas , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Suelo , Agua/metabolismoRESUMEN
Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both "hot and cold" regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.
Asunto(s)
Arabidopsis/genética , Eucromatina/genética , Heterocromatina/genética , Recombinación Homóloga/genética , Arabidopsis/crecimiento & desarrollo , Sistemas CRISPR-Cas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Genoma de Planta/genética , Reparación del ADN por Recombinación/genéticaRESUMEN
The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
RESUMEN
Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.
