植物科学

Rice (Oryza sativa), a staple crop sustaining half of humanity’s caloric intake, is threatened by numerous insect-vector-transmitted diseases, such as rice stripe disease, caused by the rice stripe virus (RSV). Most genetic studies on plant antiviral defense mechanisms rely on natural or artificial infection and transgenic approaches, which require months of plant transformation. Here, we present a streamlined protocol that enables rapid analysis of RSV–host interactions within three days. The method encompasses three key phases: (1) polyethylene glycol (PEG)-based precipitation of RSV virions from infected plant tissues, (2) sequential purification through differential ultracentrifugation with glycerol cushion optimization, and (3) high-efficiency transfection of purified virions into rice protoplasts via PEG-mediated delivery. Viral replication is quantitatively assessed using RT-qPCR targeting viral RNA and immunoblotting with RSV nucleocapsid protein-specific monoclonal antibodies. This approach eliminates dependency on stable transgenic lines, allowing the simultaneous introduction of exogenous plasmids for functional studies. Compared with conventional methods requiring several months for transgenic plant generation, our protocol delivers analyzable results within three days, significantly accelerating the exploration of antiviral mechanisms and resistance gene screening.

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.

Plant growth–promoting rhizobacteria (PGPR) can be used as biofertilizers to enhance crop growth for better yield and soil fertility restoration. PGPR possesses certain traits such as nutrient solubilization, phytohormone production, and production of key enzymes for improved crop growth. These traits are also important for inhibiting the growth of plant root pathogens, improving root development, and conferring stress tolerance. However, the mere presence of PGPR traits in isolated bacteria may not directly reflect an improvement in plant growth, warranting researchers to evaluate phenotypic and physiological changes upon inoculation. The current manuscript provides a detailed step-by-step procedure for inoculating the PGPR Staphylococcus sciuri into seeds and seedlings of rice and tomato plants for visualizing the enhancement of root and shoot growth. The surface-sterilized seeds of rice and tomato plants are inoculated overnight with an actively grown log-phase culture of S. sciuri, and differences in growth and biomass of seedlings that emerged from the inoculated and uninoculated seeds are analyzed 10 days after germination. Plants grown in pots with sterile soil are also treated with PGPR S. sciuri by soil drenching. A remarkable increase in root and shoot growth is observed in inoculated plants. We suggest that treating seeds with bacteria and enriching the soil with bacterial inoculum provides an adequate load of PGPR that facilitates growth improvement. This method can be a reliable choice for screening and evaluating plant growth promotion by either isolated bacteria or bacterial consortia with plant-beneficial traits.

Most terrestrial plants are associated with symbiotic Glomeromycotina fungi, commonly known as arbuscular mycorrhizal (AM) fungi. AM fungi increase plant biomass in phosphate-depleted conditions by allocating mineral nutrients to the host; therefore, host roots actively exude various specialized metabolites and orchestrate symbiotic partners. The hyphal branching activity induced by strigolactones (SLs), a category of plant hormones, was previously discovered using an in vitro assay system. For this bioassay, AM fungi of the Gigaspora genus (Gigasporaeae) are commonly used due to their linear hyphal elongation and because the simple branching pattern is convenient for microscopic observation. However, many researchers have also used Glomeraceae fungi, such as Rhizophagus species, as the symbiotic partner of host plants, although they often exhibit a complex hyphal branching pattern. Here, we describe a method to produce and quantify the hyphal branches of the popular model AM fungus Rhizophagus irregularis. In this system, R. irregularis spores are sandwiched between gels, and chemicals of interest are diffused from the surface of the gel to the germinating spores. This method enables the positive effect of a synthetic SL on R. irregularis hyphal branching to be reproduced. This method could thus be useful to quantify the physiological effects of synthesized chemicals or plant-derived specialized metabolites on R. irregularis.

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.

Seeds ensure the growth of a new generation of plants and are thus central to maintaining plant populations and ecosystem processes. Nevertheless, much remains to be learned about seed biology and responses of germinated seedlings to environmental challenges. Experiments aiming to close these knowledge gaps critically depend on the availability of healthy, viable seeds. Here, we report a protocol for the collection of seeds from plants in the genus Populus. This genus comprises trees with a wide distribution in temperate forests and with economic relevance, used as scientific models for perennial plants. As seed characteristics can vary drastically between taxonomic groups, protocols need to be tailored carefully. Our protocol takes the delicate nature of Populus seeds into account. It uses P. deltoides as an example and provides a template to optimize bulk seed extraction for other Populus species and plants with similar seed characteristics. The protocol is designed to only use items available in most labs and households and that can be sterilized easily. The unique characteristics of this protocol allow for the fast and effective extraction of high-quality seeds. Here, we report on seed collection, extraction, cleaning, storage, and viability tests. Moreover, extracted seeds are well suited for tissue culture and experiments under sterile conditions. Seed material obtained with this protocol can be used to further our understanding of tree seed biology, seedling performance under climate change, or diversity of forest genetic resources.

Key features

• Populus species produce seeds that are small, delicate, non-dormant, with plenty of seed hair. Collection of seed material needs to be timed properly.

• Processing, seed extraction, seed cleaning, and storage using simple, sterilizable laboratory and household items only. Obtained seeds are pure, high quality, close to 100% viability.

• Seeds work well in tissue culture and in experiments under sterile conditions.

• Extractability, speed, and seed germination were studied and confirmed for Populus deltoides as an example.

• Can also serve as template for bulk seed collection from other Populus species and plant groups that produce delicate seeds (with no or little modifications).

Graphical overview

The flux in photosynthesis can be studied by performing ¹³CO₂ pulse labelling and analysing the temporal labelling kinetics of metabolic intermediates using gas or liquid chromatography linked to mass spectrometry. Metabolic flux analysis (MFA) is the primary approach for analysing metabolic network function and quantifying intracellular metabolic fluxes. Different MFA approaches differ based on the metabolic state (steady vs. non-steady state) and the use of stable isotope tracers. The main methodology used to investigate metabolic systems is metabolite steady state associated with stable isotope labelling experiments. Specifically, in biological systems like photoautotrophic organisms, isotopic non-stationary ¹³C metabolic flux analysis at metabolic steady state with transient isotopic labelling (¹³C-INST-MFA) is required. The common requirement for metabolic steady state, alongside its very short half-timed reactions, complicates robust MFA of photosynthetic metabolism. While custom gas chambers design has addressed these challenges in various model plants, no similar tools were developed for liquid photosynthetic cultures (e.g., algae, cyanobacteria), where diffusion and equilibration of inorganic carbon species in the medium entails a new dimension of complexity. Recently, a novel tailor-made microfluidics labelling system has been introduced, supplying short ¹³CO₂ pulses at steady state, and resolving fluxes across most photosynthetic metabolic pathways in algae. The system involves injecting algal cultures and medium containing pre-equilibrated inorganic ¹³C into a microfluidic mixer, followed by rapid metabolic quenching, enabling precise seconds-level label pulses. This was complemented by a ¹³CO₂-bubbling-based open labelling system (photobioreactor), allowing long pulses (minutes–hours) required for investigating fluxes into central C metabolism and major products. This combined labelling procedure provides a comprehensive fluxome cover for most algal photosynthetic and central C metabolism pathways, thus allowing comparative flux analyses across algae and plants.

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.