植物科学

Rhamnogalacturonan-II (RG-II) is one of the least studied domains of pectin, primarily due to its low abundance, the lack of reliable antibodies, and the complexity of its structure. The present study builds upon existing protocols and procedures used to analyse RG-II in tissues where it is more abundant, combining and adapting them for the isolation of RG-II from Arabidopsis seed mucilage—a structure previously thought to lack RG-II. By applying these adapted methods, we first confirmed the presence of RG-II in seed mucilage and subsequently succeeded in isolating it from a tissue where it is typically present in low abundance, thereby enabling future studies on this previously overlooked component.

Phospholipids are major structural and regulatory elements of biological membranes and are involved in many different cellular and physiological processes. In this protocol, we provide an easy, cost-effective, and efficient method to obtain an overview of the phospholipid composition using high-performance thin layer chromatography (HPTLC). While the currently known phospholipid separation methods based on HPTLC display co-migration of certain lipid classes, the method we describe here allows the separation of all phospholipid classes, including anionic phospholipids in plant samples. This protocol combines elements of the classical Vitiello and Touchstone solvent systems to optimize phospholipid separation in a scaled pattern. Here, we provide a full characterization of this method, including statistical analyses of the retention factor of each phospholipid to show the robustness of the method and its efficiency in separating all phospholipid classes of a biological sample.

Adult muscle stem cells (MuSCs) are the key cellular source for regenerating skeletal muscle in vertebrates. MuSCs are typically identified in skeletal muscle by the expression of the paired box protein 7 (PAX7) protein. Here, we developed a combined RNA fluorescent in situ hybridization (FISH) using RNAscope technology and an immunofluorescence (IF) protocol for the simultaneous detection of Pax7 mRNA and PAX7 protein in individual MuSCs in vivo. Interestingly, we show that while most PAX7⁺ (protein) MuSCs express Pax7 mRNA, there is a subset of Pax7⁺ (mRNA) cells that do not express PAX7 protein. Altogether, we developed a combined FISH/IF protocol that allows for the co-detection of mRNA and protein in MuSCs in vivo, a strategy that can be applied to any target gene. The functional significance of the Pax7-expressing subset of cells lacking PAX7 protein prior to injury remains unknown.

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.

Plant proteases participate in a wide variety of biological processes, including development, growth, and defense. To date, numerous proteases have been functionally identified through genetic studies. However, redundancy among certain proteases can obscure their roles, as single-gene loss-of-function mutants often exhibit no discernible phenotype, limiting identification through genetic approaches. Here, we describe an efficient system for the identification of target proteases that cleave specific substrates in the Arabidopsis apoplastic fluid. The method involves using Arabidopsis-submerged culture medium, which contains apoplastic proteases, followed by native two-dimensional electrophoresis. Gel fractionation and an in-gel peptide cleavage assay with a fluorescence-quenching peptide substrate are then used to detect specific proteolytic activity. The active fraction is then subjected to mass spectrometry–based proteomics to identify the protease of interest. This method allows for the efficient and comprehensive identification of proteases with specific substrate cleavage activities in the apoplast.

Cannabis (Cannabis sativa L.) derivatives are of great importance in the medical, cosmetic, and pharmaceutical industries. This relevance is mainly due to the active principles (cannabinoids) found mainly in the trichomes of the female inflorescences. One of the most commonly used methods to propagate cannabis is by vegetative stem cuttings. This low-cost technique produces genetically uniform plants, ensuring consistent growth rates and cannabinoid production. The extraction of cannabinoids and other active compounds from the resin of the flowers is the main limitation of cannabis processing. Here, we describe a step-by-step protocol for propagating female cannabis plants from vegetative stem cuttings, inducing flower development, and obtaining high-quality cannabinoid-enriched resin.

Cucumber (Cucumis sativus) trichomes play a critical role in resisting external biological and abiotic stresses. Glandular trichomes are particularly significant as they serve as sites for the synthesis and secretion of secondary metabolites, while non-glandular trichomes are pivotal for determining the appearance quality of cucumbers. However, current methods for separating trichomes encounter challenges such as low efficiency and insufficient accuracy, limiting their applicability in multi-omics sequencing studies. This protocol introduces an efficient system designed for the precise separation of glandular and non-glandular trichomes from cucumber fruit. The process begins with the pre-cooling of sorbitol buffer or ethanol solution and the RNA-free treatment of laboratory supplies, followed by sterilization and pre-cooling. After filling glass bottles with pre-cooling buffer and glass beads, cucumber ovaries are then placed in the glass bottles and the trichome is harvested by bead-beating method. The separation process involves sequential filtration through various steel sieves and centrifugation to separate trichomes. The separated trichomes obtained from this method are well-suited for subsequent multi-omics sequencing analyses. This protocol achieved high precision in separating glandular and non-glandular trichomes, significantly enhancing the efficiency of separation and sample collection processes. This advancement not only addresses existing limitations but also facilitates comprehensive studies aimed at exploring the genetic and biochemical diversity present within cucumber trichomes, thereby opening avenues for broader agricultural and biological research applications.

The target of rapamycin (TOR) is a central hub kinase that promotes growth and development in all eukaryote cells. TOR induces protein synthesis through the phosphorylation of the S6 kinase (S6K), which, in turn, phosphorylates ribosomal S6 protein (RPS6) increasing this anabolic process. Therefore, S6K and RPS6 phosphorylation are generally used as readouts of TOR activity. Protein phosphorylation levels are measured by a western blot (WB) technique using an antibody against one specific phosphosite in cell extracts. However, at the tissue/cell-specific level, there is a huge gap in plants due to the lack of alternative techniques for the evaluation of TOR activity as there are for other organisms such as mammals. Here, we describe an in vivo protocol to detect S6K phosphorylation in tissues/cells of model photosynthetic organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Our proposed method consists of the immunolocalization of a phosphorylated target of TOR kinase using a fluorescent secondary antibody by confocal microscopy. The protocol involves four main steps: tissue/cell fixation, permeabilization, and incubation with primary and secondary antibodies. It is an easy technique that allows handling different samples at the same time. In addition, different ultrastructural cell markers can also be used, such as for nucleus and cell wall detection, allowing a detailed analysis of cell morphology. To our knowledge, this is the first protocol to detect TOR activity in situ in photosynthetic organisms; we consider that it will pave the research on the TOR kinase, opening new possibilities to better understand its complex signaling.

Carbohydrates serve crucial functions in most living cells, encompassing structural and metabolic roles. Within the realms of plant and algal biology, carbohydrate biosynthesis and partitioning play pivotal roles in growth, development, stress physiology, and various practical applications. These applications span diverse fields, including the food and feed industry, bioenergetics (biofuels), and environmental management. However, existing methods for carbohydrate determination tend to be costly and time-intensive. In response to that, we propose a novel approach to assess carbohydrate partitioning from small samples. This method leverages the differential solubility of various fractions, including soluble sugars, starch, and structural polymers (such as cellulose). After fractionation, a straightforward spectrophotometric analysis allows for the quantification of sugars.

Extracellular vesicles are membrane-bound organelles that play crucial roles in intercellular communication and elicit responses in the recipient cell, such as defense responses against pathogens. In this study, we have optimized a protocol for isolating extracellular vesicles (EVs) from Sorghum bicolor apoplastic wash. We characterized the EVs using fluorescence microscopy and correlative light and electron microscopy.