Bundgaardraynor4981

Electron microscopy enables the unbiased imaging of organelles and cellular structures at nano-meter scale resolution. The combination of cryofixation/freeze-substitution methods with other imaging techniques such as correlative light and electron microscopy (CLEM), electron tomography (ET), and immunogold-labeling provides unique opportunities to understand structural changes associated with cellular processes. This chapter presents the main steps in the preparation of Arabidopsis thaliana roots, cotyledons, anthers, and developing seeds by high-pressure freezing and freeze-substitution for structural analysis and immunogold-labeling using transmission electron microscopy.The study of protein-protein interaction (PPI) is critical for understanding cellular processes within biological systems. The conventional biomolecular fluorescence complementation (BiFC) or bipartite split-fluorescent protein (FP) is a noninvasive fluorescent-based technique that enables direct visualization of PPI in living cells once the two nonfluorescent fragments are brought into close vicinity. However, BiFC can potentially lead to a high background noise arising from an inherent feature of the irreversible self-assembly of the nonfluorescent fragments. Recently, the newly developed tripartite split-sfGFP method was demonstrated to detect membrane PPIs in plant cells without spurious background signals even when fusion proteins are highly expressed and accessible to the compartments of interaction. Here we describe a protocol for using the ß-Estradiol-inducible tripartite split-sfGFP assay for side-by-side analyses of in vivo PPI along with in situ subcellular localization of fusion proteins in agroinfiltrated Nicotiana benthamiana leaves.Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)-based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca2+ indicator, MatryoshCaMP6s.Plants develop lateral organs such as leaves and flowers throughout their post-embryonic life from a structure called the shoot apical meristem (SAM), located at the plant shoot apex. This process is highly dynamic, and therefore in order to understand meristem and organ development, it is critical to be able to analyze these processes with high temporal and spatial resolution. Although several protocols have been published for imaging the Arabidopsis inflorescence meristem, gaining access to the vegetative meristem for imaging has been considered more difficult. DCZ0415 mouse Here we describe a method to dissect young Arabidopsis seedlings in order to obtain a clear view of the vegetative meristem and young leaf primordia using confocal microscopy.Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of cellular populations. Applications involving higher plants started to emerge around 40 years ago and are now widely employed both to provide unique information regarding basic and applied questions in the biosciences and to advance agricultural productivity in practical ways. Further development of this platform is being actively pursued, and this promises additional progress in our understanding of the interactions of cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types held together by tough cell walls, and, second, because individual plant cells are generally larger than those of mammals.This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. In Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in molecular biology, vol 1062. Humana Press, Totowa, pp 509-537], describes the application of techniques of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing (a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei, and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are emphasized.RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.In eukaryotes, DNA is packed into an incredibly complex structure called chromatin. Although chromatin was often considered as a static entity, it is now clear that chromatin proteins and the chromatin fiber itself are in fact very dynamic. For instance, the packaging of the DNA into the nucleus requires an extraordinary degree of compaction but this should be achieved without compromising the accessibility to the transcription machinery and other nuclear processes. Approaches such as gene tagging have been established for living cells in order to detect, track, and analyze the mobility of single loci. In this chapter, we provide an experimental protocol for performing locus tracking in Arabidopsis thaliana roots and for characterizing locus mobility behavior via a Mean Square Displacement analysis.