RESUMO
Single-cell sequencing approaches reveal the intracellular dynamics of individual cells and answer biological questions with high-dimensional catalogs of millions of cells, including genomics, transcriptomics, chromatin accessibility, epigenomics, and proteomics data across species. These emerging yet thriving technologies have been fully embraced by the field of plant biology, with a constantly expanding portfolio of applications. Here, we introduce the current technical advances used for single-cell omics, especially single-cell genome and transcriptome sequencing. Firstly, we overview methods for protoplast and nucleus isolation and genome and transcriptome amplification. Subsequently, we use well-executed benchmarking studies to highlight advances made through the application of single-cell omics techniques. Looking forward, we offer a glimpse of additional hurdles and future opportunities that will introduce broad adoption of single-cell sequencing with revolutionary perspectives in plant biology.
Assuntos
Genômica , Proteômica , Epigenômica/métodos , Genoma , Genômica/métodos , Metabolômica/métodos , Plantas/genética , Proteômica/métodos , Transcriptoma/genéticaRESUMO
Compound leaves are composed of multiple separate blade units termed leaflets. In tomato (Solanum lycopersicum) compound leaves, auxin promotes both leaflet initiation and blade expansion. However, it is unclear how these two developmental processes interact. With highly variable complexity, tomato compound leaves provide an ideal system to address this question. In this study, we obtained and analyzed mutants of the WUSCHEL-RELATED HOMEOBOX (WOX) family gene SlLAM1 from tomato, whose orthologs in tobacco (Nicotiana sylvestris) and other species are indispensable for blade expansion. We show that SlLAM1 is expressed in the middle and marginal domains of leaves, and is required for blade expansion in leaflets. We demonstrate that sllam1 mutants cause a delay of leaflet initiation and slightly alter the arrangement of first-order leaflets, whereas the overall leaflet number is comparable to that of wild-type leaves. Analysis of the genetic interactions between SlLAM1 and key auxin signaling components revealed an epistatic effect of SlLAM1 in determining the final leaf form. Finally, we show that SlLAM1 is also required for floral organ growth and affects the fertility of gametophytes. Our data suggest that SlLAM1 promotes blade expansion in multiple leaf types, and leaflet initiation can be largely uncoupled from blade expansion during compound leaf morphogenesis.
Assuntos
Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Transdução de Sinais , Solanum lycopersicum/genética , Genes Homeobox , Solanum lycopersicum/crescimento & desenvolvimento , Mutação , Filogenia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Understanding protein-protein interactions (PPIs) through proximity labeling has revolutionized our comprehension of cellular mechanisms and pathology. Various proximity labeling techniques, such as HRP, APEX, BioID, TurboID, and µMap, have been widely used to biotinylate PPIs or organelles for proteomic profiling. However, the variability in labeling precision and efficiency of these techniques often results in limited reproducibility in proteomic detection. We address this persistent challenge by introducing proximity labeling expansion microscopy (PL-ExM), a super-resolution imaging technique that combines expansion microscopy with proximity labeling techniques. PL-ExM enabled up to 17 nm resolution with microscopes widely available, providing visual comparison of the labeling precision, efficiency, and false positives of different proximity labeling methods. Our mass spectrometry proteomic results confirmed that PL-ExM imaging is reliable in guiding the selection of proximity labeling techniques and interpreting the proteomic results with new spatial information.
RESUMO
Due to the abuse of antibiotics, antibiotic residues can be detected in both natural environment and various industrial products, posing threat to the environment and human health. Here we describe the design and implementation of an engineered Escherichia coli capable of degrading tetracycline (Tc)-one of the commonly used antibiotics once on humans and now on poultry, cattle and fisheries. A Tc-degrading enzyme, TetX, from the obligate anaerobe Bacteroides fragilis was cloned and recombinantly expressed in E. coli and fully characterized, including its K m and k cat value. We quantitatively evaluated its activity both in vitro and in vivo by UV-Vis spectrometer and LC-MS. Moreover, we used a tetracycline inducible amplification circuit including T7 RNA polymerase and its specific promoter PT7 to enhance the expression level of TetX, and studied the dose-response of TetX under different inducer concentrations. Since the deployment of genetically modified organisms (GMOs) outside laboratory brings about safety concerns, it is necessary to explore the possibility of integrating a kill-switch. Toxin-Antitoxin (TA) systems were used to construct a mutually dependent host-plasmid platform and biocontainment systems in various academic and industrious situations. We selected nine TA systems from various bacteria strains and measured the toxicity of toxins (T) and the detoxifying activity of cognate antitoxins (A) to validate their potential to be used to build a kill-switch. These results prove the possibility of using engineered microorganisms to tackle antibiotic residues in environment efficiently and safely.