Plant Science

We present a protocol to allow continuous assessment of cell death in Arabidopsis thaliana (L.) seedlings by measuring the release of electrolytes from dying cells upon heat shock. The electrolyte leakage assay is a well-established method to quantify the extent of cell death of plant tissues exposed to pathogen infection, since the activation of the immune response leads to compromised membrane integrity and to the release of ions from the dying cell. This prolonged release of electrolytes is considered a hallmark of regulated cell death in plants. Heat shock in plants induces ferroptosis-like cell death, which can be suppressed either pharmacologically, using inhibitors such as ferrostatin, or genetically through knockout of ferroptosis-related genes. Here, we have adapted the electrolyte leakage assay to quantify cell death in young Arabidopsis seedlings exposed to a heat shock previously shown to induce ferroptosis-like cell death. We also illustrate how this method can be used to assess activation of ferroptosis-like cell death in whole Arabidopsis seedlings using ferrostatin or knockout mutants of potential gene candidates involved in ferroptosis-like cell death.

The protochlorophyllide (Pchlide) level is a crucial indicator of plant fitness. Precise quantification of Pchlide content is necessary not only in studies of flu-related mutants that over-accumulate Pchlide in the dark but also for research on plants suffering from environmental stresses. Due to its low content and interference of chlorophylls, quantitative determination of Pchlide content is a challenge. Here, we describe an optimized protocol for Pchlide extraction from Arabidopsis thaliana seedlings and subsequent analysis using high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Divinyl-Protochlorophyllide (DV-Pchlide, the major form of Pchlide in plants) quantification is achieved by interpolating fluorescence peak areas against an experimentally derived standard curve. This protocol provides a reliable workflow for Pchlide quantification, facilitating the deciphering of the underlying mechanism of plant environmental resilience.

Roots are essential organs for plants, facilitating water and nutrient uptake from the soil to support growth. Traditional methods for studying root systems, such as rhizoboxes and rhizotrons, have provided valuable insights. However, advanced methods such as fabricated ecosystems (EcoFAB) combined with new generation microscopes now enable a more detailed investigation of the rhizosphere, the microenvironment surrounding roots, allowing a deeper understanding of root tissue, exudates, and plant–soil interactions. This microenvironment can be used to investigate the adaptation of plants to environmental stress (salinity, drought, higher temperatures). Our procedure focuses on establishing standardized protocols for plant growth tailored to the EcoFAB system, which offers a controlled environment to study root dynamics. This work also contributes new insights into the early stages of plant germination, an area currently underexplored in the literature. While numerous studies focus on plant growth or genetic aspects, such as gene induction, the germination phase remains underexplored. We have developed optimized germination protocols for multiple plant species, ensuring uniform seedling size and sufficient development for seamless integration into the EcoFAB system.

The root meristem navigates the highly variable soil environment where water availability limits water absorption, slowing or halting growth. Traditional studies use uniform high osmotic potentials, poorly representing natural conditions where roots gradually encounter increasing osmotic potentials. Uniform high osmotic potentials reduce root growth by inhibiting cell division and shortening mature cell length. This protocol describes a simple and effective in vitro system using a gradient mixer that generates a vertical gradient in an agar gel based on the principle of communicating vessels, exploiting gravity to generate a continuous mannitol concentration gradient (from 0 to 400 mM mannitol) reaching osmotic potentials of -1,2 MPa. It enables long-term Arabidopsis root growth analysis under progressive water deficit, improving phenotyping and molecular studies in soil-like conditions.

Chlamydomonas (Chlamydomonas reinhardtii) is a unicellular model alga that has been shown to undergo programmed cell death (PCD) that can be triggered in response to different stresses. We have recently shown that Chlamydomonas is particularly well suited to the study and quantification of PCD. We have shown for the first time that S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, is able to induce PCD and can be used as a study system in Chlamydomonas. In this article, we provide a simple and robust protocol for quantifying GSNO-induced PCD, which can be adapted to any other treatment. We explain how to detect NO production in the cell following GSNO treatment. We show how PCD can be identified simply by analyzing the degradation profile of genomic DNA. We also provide an easy and reproducible cell death quantification protocol, which makes it possible to follow the course of PCD over time and highlight very fine differences in the number of affected cells between different samples.

Murashige-Skoog medium solutions have been used in a variety of plant plate growth assays, yet most research uses Arabidopsis thaliana as the study organism. For larger seeds such as maize (Zea mays), most protocols employ a paper towel roll method for experiments, which often involves wrapping maize seedlings in wet, sterile germination paper. What the paper towel roll method lacks, however, is the ability to image the roots over time without risk of contamination. Here, we describe a sterile plate growth assay that contains Murashige-Skoog medium to grow seedlings starting two days after germination. This protocol uses a section of a paper towel roll method to achieve uniform germination of maize seedlings, which are sterilely transferred onto large acrylic plates for the duration of the experiment. The media can undergo modification to include an assortment of plant hormones, exogenous sugars, and other chemicals. The acrylic plates allow researchers to freely image the plate without disturbing the seedlings and control the environment in which the seedlings are grown, such as modifications in temperature and light. Additionally, the protocol is widely adaptable for use with other cereal crops.

Key features

• Builds upon plate growth methods routinely used for Arabidopsis seedlings but that are inadequate for maize.

• Real-time photographic analysis of seedlings up to two weeks following germination.

• Allows for testing of various growth conditions involving an assortment of additives and/or modification of environmental conditions.

• Samples are able to be collected for genotype screening.

Graphical overview

The chloroplast lumen contains at least 80 proteins whose function and regulation are not yet fully understood. Isolating the chloroplast lumen enables the characterization of the lumenal proteins. The lumen can be isolated in several ways through thylakoid disruption using a Yeda press or sonication, or through thylakoid solubilization using a detergent. Here, we present a simple procedure to isolate thylakoid lumen by sonication using leaves of the plant Arabidopsis thaliana. The step-by-step procedure is as follows: thylakoids are isolated from chloroplasts, loosely associated thylakoid surface proteins from the stroma are removed, and the lumen fraction is collected in the supernatant following sonication and centrifugation. Compared to other procedures, this method is easy to implement and saves time, plant material, and cost. Lumenal proteins are obtained in high quantity and purity; however, some stromal membrane–associated proteins are released to the lumen fraction, so this method could be further adapted if needed by decreasing sonication power and/or time.

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.

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