Plant Science

The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins.

Graphical abstract

Iron (Fe) is an indispensable micronutrient for plant growth and development. Since both deficiency, as well as a surplus of Fe, can be detrimental to plant health, plants need to constantly tune uptake rates to maintain an optimum level of Fe. Quantification of Fe serves as an important parameter for analyzing the fitness of plants from different accessions, or mutants and transgenic lines with altered expression of specific genes. To quantify metals in plant samples, methods based on inductively coupled plasma-optical emission spectrometry (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS) have been widely employed. Although these methods are highly accurate, these methodologies rely on sophisticated equipment which is not always available. Moreover, ICP-OES and ICP-MS allow for surveying several metals in the same sample, which may not be necessary if only the Fe status is to be determined. Here, we outline a simple and cost-efficient protocol to quantify Fe concentrations in roots and shoots of Arabidopsis seedlings, by using a spectroscopy-based assay to quantify Fe²⁺-BPDS₃ complexes against a set of standards. This protocol provides a fast and reproducible method to determine Fe levels in plant samples with high precision and low costs, which does not depend on expensive equipment and expertise to operate such equipment.

Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H⁺ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.

The analysis of chemical diversity in non-sterile rhizosphere soil has been a pressing methodological challenge for years. Rhizosphere-enriched chemicals (i.e., rhizochemicals) include root exudation chemicals, (microbial) breakdown products thereof, and de novo produced metabolites by rhizosphere-inhabiting microbes, all of which can play an important role in plant-soil interactions. The power and resolution of analytical methods and statistical analysis pipelines allow for better acquisition, separation and identification of rhizochemicals, thus providing unprecedented insight into the biochemistry underpinning plant-soil interactions. The current protocol describes a recently developed method to characterize rhizochemical profiles from plants, including crops, and is modular and customizable, allowing for application across a range of different plant-soil combinations. The protocol provides in-depth details about the experimental system for sample collection, data acquisition by liquid chromatography coupled to mass spectrometry, and analytical pipeline, which statistically selects for rhizochemicals by statistical comparison between metabolite profiles from plant-containing soil and plant-free soil. Moreover, the optional addition of chemical standards permits a semi-targeted approach, which improves the annotation of chemical signatures and identification of single rhizochemicals.

Insect pollinators, herbivores and their natural enemies use olfactory cues emitted by their host plants to locate them. In insect-plant ecology, understanding the mechanisms underlying these interactions are of critical importance, as this bio-communication has both ecological and agricultural applications. However, the first step in such research is to identify and quantify the insect community associated with the plant/s species of interest. Traditionally, this has been accomplished by a variety of insect trapping methods, either using pitfall traps, or sticky traps, or sweep nets in field. The data collected from these traps tend to be incomplete, and also damage the specimens, making them unusable for any taxonomic purposes. This protocol derives ideas from these traditional traps and use a combination of three easily made inexpensive modified traps that conceals the host plant, but allows the plant volatiles to pass through as olfactory cues. These traps are economical, can be made to fit with most plant sizes, and are also reusable. Collectively, these traps will provide a solid estimate (quantifiable) of all associated community of arthropods that can also be stored for future studies.

Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.

Nitrate reductase (NR) reduces the major plant nitrogen source, NO₃^-, into NO₂^-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.

Silica cells are specialized leaf epidermal cells in grasses with almost the whole cell volume filled with solid silica. In sorghum, silica deposition in silica cells takes place in young, elongating leaves around the mid-length of the leaf. We developed a protocol for estimating the level of silica cell silicification in Sorghum bicolor leaves using in situ charring method (Kumar et al., 2017a). Here, we provide greater details on our protocol and method of image analysis. Although we based our protocol on sorghum, this protocol can be extended for estimating silica cell silicification level in any grass species.

Inorganic phosphorus is a non-renewable resource and an essential element for life on Earth. Organisms such as algae, protists, and animals can store phosphate (Pi) through uptake of Pi as polyphosphate (poly-P), which is a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds. Here, we describe procedures for extraction of total phosphate and poly-P from Parachlorella cells and quantification of orthophosphate based on molybdenum blue assay. The present method may be applicable for other microalgae.

Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.