Membranes are very complex and dynamic structures that are essential for plant cellular functions and whose lipidic composition can be influenced by numerous factors. Anionic phospholipids, which include phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphoinositides are key components of these membranes as they are involved in plant cell signaling and as even slight modifications in their quantities may largely impact the cell metabolism. However, the presence of these compounds in low amounts, as well as their poor stability during analysis by mass spectrometry, make their study very complicated. In addition, the precise quantification of all anionic phospholipid species is not possible by lipid separation using thin-layer chromatography followed by the analysis of their fatty acyl chains by gas chromatography. Here, we describe a straightforward strategy for the extraction and semi-quantification of all anionic phospholipid species from plant samples. Our method is based on the derivatization of the anionic phospholipids, and more especially on their methylation using trimethylsilyldiazomethane, followed by analysis by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer. This approach allows largely improving the sensitivity of the analysis of anionic phospholipids from plant samples, which will help to gain deeper insights into the functions and dynamics of these key parts of plant cellular signaling.

Understanding how multicellular organisms are shaped requires high-resolution, quantitative data to unravel how biological structures grow and develop over time. In recent years, confocal live imaging has become an essential tool providing insights into developmental dynamics at cellular resolution in plant organs such as leaves or meristems. In the context of flowers, growth tracking has primarily been limited to sepals, the outermost floral organs, or the post-fertilization gynoecium, which are easily accessible for microscopy. Here, we describe a detailed pipeline for the preparation, dissection, and confocal imaging of the development of internal reproductive floral organs of Arabidopsis thaliana including both the stamen and gynoecium. We also discuss how to acquire high-quality images suitable for efficient 2D and 3D segmentation that allow the quantification of cellular dynamics underlying their development.

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Estimating the time of most recent common ancestor (tMRCA) is important to trace the origin of pathogenic viruses. This analysis is based on the genetic diversity accumulated in a certain time period. There have been thousands of mutant sites occurring in the genomes of SARS-CoV-2 since the COVID-19 pandemic started; six highly linked mutation sites occurred early before the start of the pandemic and can be used to classify the genomes into three main haplotypes. Tracing the origin of those three haplotypes may help to understand the origin of SARS-CoV-2. In this article, we present a complete protocol for the classification of SARS-CoV-2 genomes and calculating tMRCA using Bayesian phylodynamic method. This protocol may also be used in the analysis of other viral genomes.

Key features

• Filtering and alignment of a massive number of viral genomes using custom scripts and ViralMSA.

• Classification of genomes based on highly linked sites using custom scripts.

• Phylodynamic analysis of viral genomes using Bayesian evolutionary analysis sampling trees (BEAST).

• Visualization of posterior distribution of tMRCA using Tracer.v1.7.2.

• Optimized for the SARS-CoV-2.

Graphical overview

Graphical workflow of time of most recent common ancestor (tMRCA) estimation process

Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters.

Graphical overview

A number of molecules, such as secreted peptides, have been shown to mediate root-to-shoot signaling in response to various conditions. The xylem is a pathway for water and molecules that are translocated from roots to shoots. Therefore, collecting and analyzing xylem exudates is an efficient approach to study root-to-shoot long-distance signaling. Here, we describe a step-by-step protocol for the collection of xylem exudate from the model plant Arabidopsis and the crop plant soybean (Glycine max). In this protocol, we can collect xylem exudate from plants cultured under normal growth conditions without using special equipment.

Graphical abstract:

Xylem exudates on the cut surfaces of an Arabidopsis hypocotyl and a soybean internode.

In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle.

Graphical abstract:

Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.

The functional performance of a cell depends on how macromolecules, in particular proteins, come together in a precise orientation, how they assemble into protein complexes and interact with each other. In order to study protein-protein interactions at a molecular level, a variety of methods to investigate these binding processes yield affinity constants and/or the identification of binding regions. There are several well-established biophysical techniques for biomolecular interaction studies, such as fluorescence spectroscopy and surface plasmon resonance. Although these techniques have been proven to be efficient, they either need labeling or immobilization of one interaction partner. Backscattering interferometry (BSI) is a label-free detection method, which allows label- and immobilization-free interaction analysis under physiologically relevant conditions with high sensitivity and in small volumes. We used BSI to measure the interaction of the neuronal calcium sensor recoverin with its target G protein-coupled receptor kinase 1 (GRK1) as a model system. Increasing concentrations of purified recoverin were mixed with a specific concentration of a GRK1 fusion protein. In this protocol, we provide a full description of the instrumental setup, data acquisition, and evaluation. Equilibrium dissociation constants of recoverin-GRK1 interaction determined by the BSI instrumental setup are in full agreement with affinity constants obtained by different methods as described in the literature.

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter Glt_Ph as a model system. We prepare proteo-liposomes containing reconstituted purified Glt_Ph transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.

The in vivo toxicity of new metallodrugs either as Small Bioactive Molecules (SBAMs) or Conjugates of Metals with Drugs (CoMeDs) or their hydrogels such as with hydroxyethyl-methacrylate (HEMA) (pHEMA@SBAMs or pHEMA@CoMeDs) are evaluated by the brine shrimp assay. Thus individuals of Artemia salina larvae are incubated in saline solutions with SBAMs, CoMeDs, pHEMA@SBAMs or pHEMA@CoMeDs or without for 24 h. The toxicity is then determined in terms of the mortality rate of brine shrimp larvae. Brine shrimp assay is a low cost, safe, no required feeding during the assay, while it requiring only a small amount of the tested agent.