植物科学

In response to environmental changes, chloroplasts, the cellular organelles responsible for photosynthesis, undergo intracellular repositioning, a phenomenon known as chloroplast movement. Observing chloroplast movement within leaf tissues remains technically challenging in leaves consisting of multiple cell layers, where light scattering and absorption hinder deep tissue visualization. This limitation has been particularly problematic when analyzing chloroplast movement in the mesophyll cells of C₄ plants, which possess two distinct types of concentrically arranged photosynthetic cells. In response to stress stimuli, mesophyll chloroplasts aggregate toward the inner bundle sheath cells. However, conventional methods have not been able to observe these chloroplast dynamics over time in living cells, making it difficult to assess the influence of adjacent bundle sheath cells on this movement. Here, we present a protocol for live leaf section imaging that enables long-term and detailed observation of chloroplast movement in internal leaf tissues without chemical fixation. In this method, a leaf blade section prepared either using a vibratome or by hand was placed in a groove made of a silicone rubber sheet attached to a glass slide for microscopic observation. This technique allows for the quantitative tracking of chloroplast movement relative to the surrounding cells. In addition, by adjusting the sectioning angle and thickness of the unfixed leaf sections, it is possible to selectively inactivate specific cell types based on their size and shape differences. This protocol enables the investigation of the intercellular interactions involved in chloroplast dynamics in leaf tissues.

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.

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

In many plant species, self-incompatibility (SI) is a mechanism that inhibits inbreeding. SI is gametophytic in the Solanaceae, with specificity determined by S-ribonucleases (S-RNases) in the pistil and S-locus F-box proteins (SLFs) in the pollen. The role of these proteins has been studied extensively in the Solanaceae, often using Petunia as a model. Using degenerate PCR and Sanger sequencing, this protocol identified three SLF sequences from self-incompatible diploid potato (Solanum okadae). While SLFs are well-characterized in model species like Petunia, there is limited sequence data and no standardized protocols for identifying SLFs in non-model species such as S. okadae, hindering broader insights into SI across the Solanaceae. This protocol fills that gap by using degenerate PCR and Sanger sequencing with primers designed from conserved Petunia SLF regions to identify SLF sequences in S. okadae. SLF sequences from 10 distinct Solanaceae members sharing maximum identity with the S2-haplotype of Petunia were used to design two pairs of primers targeting different regions of the target sequence. PCR amplification using designed degenerate primers yielded amplicons that were directly sequenced and joined together to get the partial SLF sequence. It was observed that the S. okadae shared an orthologous relation with the Petunia SLF according to the phylogenetic analysis. These SLFs could be used in future SI breakdown experiments via the competitive interaction route. This protocol, including the primer design, is novel for detecting SLF sequences in S. okadae.

Amyloplasts, non-photosynthetic plastids specialized for starch synthesis and storage, proliferate in storage tissue cells of plants. To date, studies of amyloplast replication in roots and the ovule nucelli from various plant species have been performed using electron and fluorescence microscopy. However, a complete understanding of amyloplast replication remains unclear due to the absence of experimental systems capable of tracking their morphology and behavior in living cells. Recently, we demonstrated that Arabidopsis ovule integument could provide a platform for live-cell imaging of amyloplast replication. This system enables precise analysis of amyloplast number and shape, including the behavior of stroma-filled tubules (stromules), during proplastid-to-amyloplast development in post-mitotic cells. Here, we provide technical guidelines for observing and quantifying amyloplasts using conventional fluorescence microscopy in wild-type and several plastid-division mutants of Arabidopsis.

In live-cell imaging, autofluorescence is often regarded as a negative factor that interferes with the accurate visualization of target fluorescence due to a phenomenon known as crosstalk. However, autofluorescence has also been effectively utilized as an organellar marker. For instance, the intense autofluorescence of chlorophyll in the red wavelength is widely used to visualize chloroplasts, the photosynthetic organelle in plants. Recently, we demonstrated that nuclei in plant cells emit phytochrome-derived autofluorescence in the red to infrared wavelength range, which can be visualized by a conventional confocal microscope equipped with a 640 nm laser. Here, we present protocols for growing plants and conducting confocal imaging of the near-infrared autofluorescence of nuclei in Arabidopsis thaliana.

Membranes are very complex and dynamic structures that are essential for plant cellular functions and whose lipidic composition can be influenced by numerous factors. Anionic phospholipids, which include phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphoinositides are key components of these membranes as they are involved in plant cell signaling and as even slight modifications in their quantities may largely impact the cell metabolism. However, the presence of these compounds in low amounts, as well as their poor stability during analysis by mass spectrometry, make their study very complicated. In addition, the precise quantification of all anionic phospholipid species is not possible by lipid separation using thin-layer chromatography followed by the analysis of their fatty acyl chains by gas chromatography. Here, we describe a straightforward strategy for the extraction and semi-quantification of all anionic phospholipid species from plant samples. Our method is based on the derivatization of the anionic phospholipids, and more especially on their methylation using trimethylsilyldiazomethane, followed by analysis by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer. This approach allows largely improving the sensitivity of the analysis of anionic phospholipids from plant samples, which will help to gain deeper insights into the functions and dynamics of these key parts of plant cellular signaling.

The ability to efficiently screen plant pathogen effectors is crucial for understanding plant–pathogen interactions and developing disease-resistant crops. Traditional methods are often labor-intensive and time-consuming. Here, we present a robust, high-throughput screening assay using the tobacco mosaic virus–green fluorescent protein (TMV-GFP) vector system. The screening system combines the TMV-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity (both activation and suppression). The biological function of these effectors can be easily evaluated within six days by observing the GFP fluorescence signal using a UV lamp. This protocol significantly reduces the time required for screening and increases the throughput, making it suitable for large-scale studies. The method is versatile, cost-effective, and can be adapted to effectors with immune interference activity from various pathogens.

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.