0 Q&A 766 Views May 20, 2024

Agrobacterium-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death.

0 Q&A 597 Views May 5, 2024

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find “false” enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements.

0 Q&A 1145 Views Sep 5, 2023

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids formed through liquid–liquid phase separation (LLPS). These assemblies are known to temporally and spatially regulate numerous biological activities and cellular processes in plants and animals. In vitro phase separation assay using recombinant proteins represents one of the standard ways to examine the properties of proteins undergoing LLPS. Here, we present a detailed protocol to investigate in vitro LLPS using in vitro expressed and purified recombinant proteins.

0 Q&A 556 Views Mar 5, 2023

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.

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0 Q&A 989 Views Nov 5, 2022

Cytochrome P450 reductase (CPR) is a multi-domain protein that acts as a redox partner of cytochrome P450s. The CPR contains a flavin adenine dinucleotide (FAD)–binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting domain. To achieve catalytic events, the FMN-binding domain needs to move relative to the FAD-binding domain, and this high flexibility complicates structural determination in high-resolution by X-ray crystallography. Here, we demonstrate a seeding technique of sorghum CPR crystals for resolution improvement, which can be applied to other poorly diffracting protein crystals. Protein expression is completed using an E. coli cell line with a high protein yield and purified using chromatography techniques. Crystals are screened using an automated 96-well plating robot. Poorly diffracting crystals are originally grown using a hanging drop method from successful trials observed in sitting drops. A macro seeding technique is applied by transferring crystal clusters to fresh conditions without nucleation to increase crystal size. Prior to diffraction, a dehydration technique is applied by serial transfer to higher precipitant concentrations. Thus, an increase in resolution by 7 Å is achieved by limiting the inopportune effects of the flexibility inherent to the domains of CPR, and secondary structures of SbCPR2c are observed.

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2 Q&A 1743 Views Oct 20, 2022

The ascorbate peroxidase (APX) is a widely distributed antioxidant enzyme. It differs from catalase and other peroxidases in that it scavenges/reduces reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) to water using reduced ascorbate as the electron donor. It is advantageous over other similar antioxidant enzymes in scavenging ROS since ascorbate may react with superoxide, singlet oxygen, and hydroxyl radical, in addition to reacting with H2O2. The estimation of its activity is helpful to analyze the level of oxidative stress in living systems under stressful conditions. The present protocol was performed to analyze the impact of heavy metal chromium (Cr) toxicity on sorghum plants in the form of APX enzyme activity under the application of glycine betaine (GB) and arbuscular mycorrhizal fungi (AMF) as stress ameliorators. Plant defense strategies against heavy metals toxicity involve the utilization of APX and the instigation of AMF symbiotic system, as well as their possible collaboration with one another or with the plant antioxidant system; this has been examined and discussed in literature. In this protocol, an increased APX activity was observed on underlying functions and detoxification capabilities of GB and AMF that are typically used by plants to enhance tolerance to Cr toxicity.

Graphical abstract:

Flow chart of standardized or calibrated enzyme assay with leaf samples of sorghum

0 Q&A 1495 Views Oct 5, 2022

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.

0 Q&A 1374 Views Oct 5, 2022
The quantification of plant hormones and related gene expression is essential to improve the understanding of the molecular regulation of plant growth and development. However, plant hormone quantification is still challenging due to extremely low endogenous levels and high chemical diversity. In this study, we present a convenient extraction protocol that enables the simultaneous extraction of both phytohormones and RNA from the same sample in a small quantity (approximately 10 mg). Using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS), this protocol provides a method to quantify 13 phytohormones and their derivatives from four classes (cytokinin, auxin, abscisic acid, and gibberellin) at the speed of 14 min per sample.

0 Q&A 2046 Views Jun 20, 2022

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor of numerous enzymatic reactions found in all living cells. Pyridine nucleotides (NAD+ and NADH) are also key players in signaling through reactive oxygen species (ROS), being crucial in the regulation of both ROS-producing and ROS-consuming systems in plants. NAD content is a powerful modulator of metabolic integration, protein de-acetylation, and DNA repair. The balance between NAD oxidized and reduced forms, i.e., the NADH/NAD+ ratio, indicates the redox state of a cell, and it is a measurement that reflects the metabolic health of cells. Here we present an easy method to estimate the NAD+ and NADH content enzymatically, using alcohol dehydrogenase (ADH), an oxido-reductase enzyme, and with MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as the substrate and 1-methoxy PMS (1-Methoxy-5-methylphenazinium methyl sulfate) as the electron carrier. MTT is reduced to a purple formazan, which is then detected. We used Arabidopsis leaf samples exposed to aluminum toxicity and under untreated control conditions. NADH/NAD+ connects many aspects of metabolism and plays vital roles in plant developmental processes and stress responses. Therefore, it is fundamental to determine the status of NADH/NAD+ under stress.

0 Q&A 1338 Views Jun 5, 2022

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation–based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes.

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