Plant Science

Paraquat is a cost-effective herbicide, widely used in many countries, that can induce severe oxidative stress in photosynthetic tissues. Studying plant herbicide resistance or antioxidant stress mechanisms requires determining the cellular paraquat level when plants are treated by paraquat. The traditional isotopic labeling method has the potential risk to cause problems to both human health and the environment. For radioisotope manipulation, special operation spaces and strict environmental inspection are also required. In addition, the radiolabeled paraquat is increasingly hard to buy due to the extended production cycle. Here, we describe a nonradioactive method to determine the paraquat level in a small number of Arabidopsis tissues or protoplasts, using a high resolution ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)/MS method. This method is highly selective and sensitive, and more environmentally compatible and technically feasible than the isotope detection method.

Glycerol-3-phosphate (G3P) is a conserved precursor of glycerolipids that also plays an important role in plant defense. Its levels and/or metabolism are also associated with many human disorders including insulin resistance, diabetes, obesity, and cancer, among others. In plants, G3P accumulates upon pathogen infection and is a critical component of systemic acquired resistance, which confers broad spectrum disease resistance against secondary infections. G3P also plays an important role in root-shoot-root signaling in soybean that regulates incompatible interactions with nitrogen-fixing bacteria. Thus, accurate quantification of G3P is key to drawing a valid conclusion regarding its role in diverse processes ranging from lipid biosynthesis to defense. G3P quantification is further compounded by its rapid degradation in extracts prepared at room temperature.

Here, we describe a simplified procedure for accurate quantitative analysis of G3P from plant tissues. G3P was extracted along with the internal standard ribitol, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by gas chromatography–coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient, and does not involve isotopic internal standards or multiple-step derivatizations.

Ethylene is an important plant hormone that is involved in the regulation of numerous processes in plant development. It also acts as a signaling molecule in response to biotic and abiotic stress conditions. Most studies have investigated ethylene evolution of harvested fruit or small herbaceous plants under controlled conditions, but only a few explored ethylene release in other plant tissues, such as leaves and buds, particularly those of subtropical crops. However, in light of increasing environmental challenges in agriculture (such as temperature extremes, droughts, floods, and high solar radiation), studies on these challenges and on potential chemical treatments for mitigating their effects on plant physiology have become more and more important. Thus, adequate techniques for the sampling and analysis of tree crops are needed to ensure accurate ethylene quantification. As part of a study on ethephon as a mitigating agent to improve litchi flowering under warm winter conditions, a protocol was developed for ethylene quantification in leaf and bud tissue of litchi following ethephon application, taking into account that these plant organs release lower ethylene concentrations than fruit. At sampling, leaves and buds were placed in glass vials of appropriate sizes for the respective plant tissue volumes and allowed to equilibrate for 10 min to release possible wound ethylene before incubating the samples for 3 h at ambient temperature. Thereafter, ethylene samples were aspirated from the vials and analyzed using a gas chromatograph with flame ionization detection, the TG-BOND Q+ column for separation of ethylene, and helium as the carrier gas. Quantification was achieved based on a standard curve derived from an external standard gas calibration with certified ethylene gas. This protocol will also be appropriate for other tree crops with similar plant materials as study foci. It will enable researchers to accurately determine ethylene production in various studies investigating the role of ethylene in general plant physiology or stress-induced plant responses following a range of treatment conditions.

Based on the availability of oxygen, plant growth environment can be normoxic (normal environment), hypoxic (reduced oxygen, <21%), or anoxic (complete depletion of oxygen). Hypoxic/anoxic environment is created when a plant is exposed to stresses such as submergence, flooding, or pathogen attack. Survival of the plants following stress conditions is in part dependent on their ability to overcome the stress induced by anoxia/hypoxia conditions. This shows the need for the development of strategies for understanding the mechanisms involved in plant tolerance to anoxia. Previous studies have employed different methods for establishing an anerobic environment. Here, we describe a simple method for creating anoxic environment using an anaerobic atmosphere generation bag. Anoxic conditions can be maintained in a cylindrical jar, a rectangular box, or a vacuum sealer bag, enabling the screening of a large number of samples. This protocol is particularly useful to screen plant mutants that are tolerant to anoxia. The method is simple, easy, cost-efficient, reproducible, and does not require any sophisticated instruments.

Graphic abstract

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana, which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues.

Graphical abstract

Identifying genetic variations or treatments that confer greater resistance to drought is paramount to ensuring sustainable crop productivity. Accurate and reproducible measurement of drought stress symptoms can be achieved via automated, image-based phenotyping. Many phenotyping platforms are either cost-prohibitive, require specific technical expertise, or are simply more complex than necessary to effectively evaluate drought resistance. Certain mutations, allelic variations, or treatments result in plants that constitutively use less water. To accurately identify genetic differences or treatments that confer a drought phenotype, plants from all experimental groups must be subjected to equal levels of drought stress. This can be easily achieved by growing and imaging plants that are grown in the same pot. Here, we provide a detailed protocol to configure a Raspberry Pi computer and camera module to image seedlings of multiple genotypes growing in shared pots and to transfer images and metadata via the cloud for downstream analyses. Also detailed is a method to calculate percent soil water content of pots while being imaged to allow for comparison of stress symptoms with water availability. This protocol was recently used to uncouple differential water usage from drought resistance in a dwarf Arabidopsis thaliana mutant chiquita1-1/cost1 compared to the wild-type control. It is cost effective, suitable for any plant species, customizable to various biological questions, and requires no prior experience with electronics or basic software programming.

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

Weeds compete with crops for growth resources, causing tremendous yield losses. Paraquat is one of the three most common non-selective herbicides. To study the mechanisms of paraquat resistance, we need to trace the movement of paraquat in plants and within the cell. ¹⁴C is a radioactive carbon isotope widely used to trace substances of interest in various biological studies, especially in transport analyses. Here, we describe a detailed protocol using ¹⁴C-paraquat to demonstrate paraquat efflux in Arabidopsis protoplasts.

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.