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Abstract Histone post‐translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP‐seq) is a powerful tool to profile histone PTMsin vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)–based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high‐quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high‐yielding histone extraction and purification method optimized forArabidopsis thalianathat can be used to obtain high‐quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS‐ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nuclear isolation and histone acid extraction Basic Protocol 2: Peptide labeling, digestion, and desalting Basic Protocol 3: Histone HPLC‐MS/MS and data analysis
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Isolation of Extracellular Vesicles from Arabidopsis
Abstract Extracellular vesicles (EVs) in plants have emerged as key players in cell‐to‐cell communication and cross‐kingdom RNAi between plants and pathogens by facilitating the exchange of RNA, proteins, and other molecules. In addition to their role in intercellular communication, plant EVs also show promise as potential therapeutics and indicators of plant health. However, plant EVs exhibit significant heterogeneity in their protein markers, size, and biogenesis pathways, strongly influencing their composition and functionality. While mammalian EVs can be generally classified as exosomes that are derived from multivesicular bodies (MVBs), microvesicles that are shed from the plasma membrane, or as apoptotic bodies that originate from cells undergoing apoptosis, plant EVs remain poorly studied in comparison. At least three subclasses of EVs have been identified inArabidopsisleaves to date, including Tetraspanin‐positive exosomes derived from MVBs, Penetration 1 (PEN1)‐positive EVs, and EVs derived from exocyst‐positive organelles (EXPO). Differences in the plant starting material and isolation techniques have resulted in different purities, quality, and compositions of the resulting EVs, complicating efforts to better understand the role of these EVs in plants. We performed a comparative analysis on commonly used plant EV isolation methods and have identified an effective protocol for extracting clean apoplastic washing fluid (AWF) and isolating high‐quality intact and pure EVs ofArabidopsis thaliana. These EVs can then be used for various applications or studied to assess their cargos and functionality in plants. Furthermore, this process can be easily adapted to other plant species of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of EVs from the apoplastic fluid ofArabidopsis thaliana Basic Protocol 2: Density gradient fractionation of EVs Basic Protocol 3: Immuno‐isolation of EVs usingArabidopsistetraspanin 8 (TET8) antibody
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- Award ID(s):
- 2020731
- PAR ID:
- 10370512
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 2
- Issue:
- 1
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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