Current protocols in plant biology最新文献

The ability to sequence DNA retrieved from ancient and historical material plays a crucial role in reinforcing evolutionary and anthropological inference. While the focus of the field is largely on analyzing DNA from ancient hominids and other animals, we have also learned from plant ancient DNA (aDNA), in particular, about human farming practices, crop domestication, environment management, species invasion, and adaptation to various environmental conditions. In the following protocols, we outline best practices for plant aDNA isolation, preparation for sequencing, bioinformatic processing, and authentication. We describe the process all the way from processing of archaeological or historical plant material to characterizing and authenticating sequencing reads. In alternative protocols, we include modifications to this process that are tailored to strongly degraded DNA. Throughout, we stress the importance of precautionary measures to successfully analyze aDNA. Finally, we discuss the evolution of the archaeogenomics field and the development of new methods, which both shaped this protocol. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of aDNA

Alternate Protocol 1: Isolation of ultra-short DNA (Dabney modification)

Support Protocol 1: Preparation of PTB-based mix

Support Protocol 2: Preparation of binding buffer

Basic Protocol 2: Preparation of genomic libraries

Alternate Protocol 2: Preparation of genomic libraries with uracil removal

Basic Protocol 3: Bioinformatic processing and authentication of aDNA

The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor-intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics’ Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid-sequencing libraries (sNucRNA-seq). © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Arabidopsis seed sterilization and planting

Basic Protocol 2: Nuclei isolation from Arabidopsis roots

Basic Protocol 3: Fluorescent-activated nuclei sorting (FANS) purification

Support Protocol: Estimation of nuclei density using Countess II automated cell counter

Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor

Alternate Protocol 2: Estimation of nuclei density using sNucRNA-seq technology

Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of Arabidopsis seedling materials

Basic Protocol 2: Isolation and enrichment of plasma membrane proteins

Support Protocol 1: Determination of protein concentration using BCA assay

Basic Protocol 3: Enrichment of S-acylated proteins by acyl-biotin exchange method

Support Protocol 2: Protein precipitation by methanol/chloroform method

Basic Protocol 4: Trypsin digestion and proteomic analysis

Alternate Protocol: Pre-resin digestion and peptide-level enrichment

Stress granules (SGs) are ubiquitous nonmembrane-bound assemblies of protein and mRNA formed under stress conditions associated with stalled translation. SGs are evolutionarily conserved across eukaryotes. The canonical function of SGs is to selectively protect mRNAs and proteins from unfolding and prevent degradation induced by diverse environmental stresses. Moreover, sequestration into SGs provides an elegant way to regulate protein activities. Disassembly of SGs upon stress recovery is accompanied by the reactivation of protein translation and protein activities. The regulatory importance of SGs has been corroborated by recent studies describing the multiomics analysis of the composition of SGs from yeast, animal, and plant cells. Herein, we describe an isolation protocol of SGs that allows for the identification of proteins, mRNA, and metabolites sequestered into SG cores. Furthermore, the described protocols can be used to isolate other SG-like foci. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of SG-enriched fraction from plant material Basic Protocol 2: Affinity purification to isolate SGs Basic Protocol 3: Simultaneous extraction of proteins and metabolites from affinity-purified beads Basic Protocol 4: Protein digestion on affinity-purified beads Basic Protocol 5: Data analysis.

Plant wax lipid molecules, chiefly normal (n-) alkanes and n-alkanoic acids, are frequently used as proxies for understanding paleoenvironmental and paleoclimatic change. These are regularly analyzed from marine and lake sediments and even more frequently in archaeological contexts, enabling the reconstruction of past environments in direct association with records of past human behavior. Carbon and hydrogen isotope measurements of these compounds are used to trace plant type and water-use efficiency, relative paleotemperature, precipitation, evapotranspiration of leaf and soil moisture, and other physiological and ecological parameters. Plant wax lipids have great potential for answering questions related to human-environment interactions, being for the most part chemically inert and easily recoverable in terrestrial sediments, including those dating back millions of years. The growing use of this technique, and comparison of such data with other paleoenvironmental proxies such as pollen and phytolith analysis and soil carbonate and tooth enamel isotope records, make it essential to establish consistent, best-practice protocols for extracting n-alkanes and n-alkanoic acids from archaeological sediments to provide comparable information for interpreting past climatic, ecosystem, and hydrological changes and their interaction with human societies. © 2020 The Authors. Basic Protocol 1: Total lipid extraction Support Protocol 1: Weighing the total lipid extract Support Protocol 2: Cleaning the PSE extraction cells Alternate Protocol 1: Soxhlet total lipid extraction Alternate Protocol 2: Ultrasonic total lipid extraction Basic Protocol 2: Separation of lipids by aminopropyl column chromatography Basic Protocol 3: Separation of lipids by silver-nitrate-infused silica gel column chromatography Support Protocol 3: Preparation of silica gel infused with 10% silver nitrate Basic Protocol 4: Methylation of n-alkanoic acids Basic Protocol 5: Gas chromatography mass spectrometry (GC-MS) Basic Protocol 6: Gas chromatography isotope ratio mass spectrometry (GC-IRMS).

CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.

Root vascular pathogens are some of the world's most devastating plant pathogens. However, the methods used to determine plant susceptibility to this class of pathogen are laborious, variable, and in most cases qualitative. Here we present a rapid, simple, and robust infection assay for the characterization of Arabidopsis thaliana resistance to the fungal root pathogen Fusarium oxysporum. The method utilizes fungal root vascular penetrations and fungal-induced root growth inhibition to deliver a quantitative assessment of plant susceptibility with spatial and temporal resolution. These plant susceptibility indicators are paired with a semiautomated data analysis pipeline to deliver a reproducible assessment of plant susceptibility to root vascular pathogens such as F. oxysporum. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis thaliana plate infection assay using fluorescently labeled Fusarium oxysporum Support Protocol 1: Preparation of A. thaliana germination plates Support Protocol 2: Preparation of the F. oxysporum culture Basic Protocol 2: Data acquisition of F. oxysporum plant infection assay Support Protocol 3: Acquiring root growth inhibition data using Fiji.

Elevation of the cytosolic free calcium ion (Ca²⁺ ) concentration ([Ca²⁺ ]_cyt ) is one of the earliest responses to biotic and abiotic stress in plant cells. Among the various Ca²⁺ detection systems available, aequorin-based luminescence Ca²⁺ imaging systems provide a relatively amenable and robust method that facilitates large-scale genetic-mutant screening based on [Ca²⁺ ]_cyt responses. Compared to that mediated by chemical elicitors, mechanical stimulation-induced elevation of [Ca²⁺ ]_cyt is considerably more rapid, occurring within 10 s following stimulation. Therefore, its assessment using aequorin-based Ca²⁺ imaging systems represents a notable challenge, given that a time interval of ≥1 min is required to reduce the background light before operating the photon imaging detector. In this context, we designed a device that can rotate automatically within the confines of an enclosed dark box, and using this, we can record [Ca²⁺ ]_cyt dynamics immediately after plants had been rotated to induce mechanical stimulation. This tool can facilitate the study of perception and early signal transduction in response to mechanical stimulation on a large scale based on [Ca²⁺ ]_cyt responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of background luminance signals in aequorin-transformed Arabidopsis seedlings using a photon imaging detector Basic Protocol 2: Construction of the rotatory device Basic Protocol 3: Calcium measurement in Arabidopsis seedlings after rotatory stimulation Basic Protocol 4: Data analysis and processing.

As the principal co-factors of many metabolic pathways, the measurement of both adenine nucleotides and nicotinamide adenine dinucleotide provides important information about cellular energy metabolism. However, given their rapid and reversible conversion as well as their relatively low concentration ranges, it is difficult to measure these compounds. Here, we describe a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides in plants. In addition, nicotinamide adenine dinucleotide is a crucially important redox-active substrate for multiple catabolic and anabolic reactions with the ratios of NAD⁺ /NADH and NADP⁺ /NADPH being suggested as indicators of the general intracellular redox potential and hence metabolic state. Here, we describe highly sensitive enzyme cycling-based colorimetric assays (with a detection limit in the pmol range) performed subsequent to a simple extraction procedure involving acid or base extraction to allow the measurement of the cellular levels of these metabolites. © 2020 The Authors. Basic Protocol 1: Preparation of plant material for the measurement Basic Protocol 2: Measurement of ATP, ADP, and AMP via HPLC Basic Protocol 3: NAD⁺ /NADP⁺ measurements Basic Protocol 4: NADH/NADPH measurements Basic Protocol 5: Data analysis and quality control approaches.