Plant Science

Chloroplast movement has been observed and analyzed since the 19^th century. Subsequently, the phenomenon is widely observed in various plant species such as fern, moss, Marchantia polymorpha, and Arabidopsis. However, chloroplast movement in rice is less investigated, presumably due to the thick wax layer on its leaf surface, which reduces light sensitivity to the point that it was previously believed that there was no light-induced movement in rice. In this study, we present a convenient protocol suitable for observing chloroplast movement in rice only by optical microscopy without using special equipment. It will allow researchers to explore other signaling components involved in chloroplast movement in rice.

Autophagy is an evolutionarily conserved intracellular degradation process. During autophagy, a set of autophagy-related (ATG) proteins orchestrate the formation of double-bound membrane vesicles called autophagosomes to engulf cytoplasmic material and deliver it to the vacuole for breakdown. Among ATG proteins, the ATG8 is the only one decorating mature autophagosomes and therefore is regarded as a bona fide autophagic marker; colocalization assays with ATG8 are wildly used as a reliable method to identify the components of autophagy machinery or autophagic substrates. Here, we describe a colocalization assay with fluorescent-tagged ATG8 using a tobacco (Nicotiana benthamiana)-based transient expression system.

Eukaryotic cells use a diverse set of transporters to control the movement of lipids across their plasma membrane, which drastically affects membrane properties. Various tools and techniques to analyze the activity of these transporters have been developed. Among them, assays based on fluorescent phospholipid probes are particularly suitable, allowing for imaging and quantification of lipid internalization in living cells. Classically, these assays have been applied to yeast and animal cells. Here, we describe the adaptation of this powerful approach to characterize lipid internalization in plant roots and aerial tissues using confocal imaging.

Graphic abstract:

Fluorescent lipid uptake in Arabidopsis seedlings. Scale bars: seedling, 25 mm; leaf, 10 μm; root, 25 μm.

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers.

Graphic abstract:

Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

In plants, the morphological diversity of leaves is largely determined by cell division, especially cell division orientation. Whereas cell division itself is easily monitored, the detection and quantification of cell division orientation are difficult. The few existing methods for detection and quantification of cell division orientation are either inefficient or laborious. Here, we describe a pulse-chase strategy using a 5-ethynyl-2’-deoxyuridine (EdU) labeling assay. Plant tissues are first incubated with EdU for a short period (pulse), followed by a long incubation without EdU (chase). Using this method, the positions of daughter cells are easily detected and can be used to quantify cell division orientation. Our protocol is rapid and very efficient for quantitative analysis of cell division orientation, and can be applied to both model and non-model plant species.

Graphic abstract


Plant cell division pairs clearly visualized by a pulse-chase EdU method

The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these essential properties of PCW, we need an appropriate tool for the accurate observation of the native (in muro) structure of the cell wall components. The label-free techniques, such as AFM, EM, FTIR, and Raman microscopy, are used; however, they either do not have the chemical or spatial resolution. Immunolabeling with electron microscopy allows observation of the cell wall nanostructure, however, it is mostly limited to single and, less frequently, multiple labeling. Immunohistochemistry (IHC) is a versatile tool to analyze the distribution and localization of multiple biomolecules in the tissue. The subcellular resolution of chemical changes in the cell wall component can be observed with standard diffraction-limited optical microscopy. Furthermore, novel chemical imaging tools such as multicolor 3D dSTORM (Three-dimensional, direct Stochastic Optical Reconstruction Microscopy) nanoscopy makes it possible to resolve the native structure of the cell wall polymers with nanometer precision and in three dimensions.

Here we present a protocol for preparing multi-target immunostaining of the PCW components taking as example Arabidopsis thaliana, Star fruit (Averrhoa carambola), and Maize thin tissue sections. This protocol is compatible with the standard confocal microscope, dSTORM nanoscope, and can also be implemented for other optical nanoscopy such as STED (Stimulated Emission Depletion Microscopy). The protocol can be adapted for any other subcellular compartments, plasma membrane, cytoplasmic, and intracellular organelles.

Live cell imaging has tremendously promoted our understanding of cellular and subcellular processes such as cell division. Here, we present a step-by-step protocol for a robust and easy-to-use live cell imaging approach to study male meiosis in the plant Arabidopsis thaliana as recently established. Our method relies on the concomitant analysis of two reporter genes that highlight chromosome configurations and microtubule dynamics. In combination, these reporter genes allowed the discrimination of five cellular parameters: cell shape, microtubule array, nucleus position, nucleolus position, and chromatin condensation. These parameters can adopt different states, e.g., the nucleus position can be central or lateral. Analyzing how tightly these states are associated gives rise to landmark stages that in turn allow a quantitative and qualitative dissection of meiotic progression. We envision that such an approach can also provide valuable criteria for the analysis of cell differentiation processes outside of meiosis.

Autophagy is the main catabolic process in eukaryotes and plays a key role in cell homeostasis. In vivo measurement of autophagic activity (flux) is a powerful tool for investigating the role of the pathway in organism development and stress responses. Here we describe a significant optimization of the tandem tag assay for detection of autophagic flux in planta in epidermal root cells of Arabidopsis thaliana seedlings. The tandem tag consists of TagRFP and mWasabi fluorescent proteins fused to ATG8a, and is expressed in wildtype or autophagy-deficient backgrounds to obtain reporter and control lines, respectively. Upon autophagy activation, the TagRFP-mWasabi-ATG8a fusion protein is incorporated into autophagosomes and delivered to the lytic vacuole. Ratiometric quantification of the low pH-tolerant TagRFP and low pH-sensitive mWasabi fluorescence in the vacuoles of control and reporter lines allows for a reliable estimation of autophagic activity. We provide a step by step protocol for plant growth, imaging and semi-automated data analysis. The protocol presents a rapid and robust method that can be applied for any studies requiring in planta quantification of autophagic flux.

Cell membrane prevents the entrance of extra molecules (e.g., transcription and translation inhibitors) into the cell. For studying the physiological effects of transcription and translation inhibitors on Hymenophyllum caudiculatum fronds, we incubate fronds with 0.1% DMSO to test if this increases cell membrane permeability relative to incubation with ultrapure water. The study showed that DMSO could significantly improve the cell membrane permeability of filmy fronds.

Filmy ferns can desiccate and recover after rehydration to resume photosynthesis. Slow and fast desiccation rates were compared in filmy fern fronds to determine whether structural or physiological differences may occur between desiccation rates. Slow desiccation is considered to be more similar to natural conditions experienced by plants that grow under the forest canopy. A fast desiccation rate will help to understand whether slow desiccation is important for recovery and viability.