We describe a user-optimized sample holder EasyClick for medium-sized plants that reduces root side movements and thus greatly extends the duration of live cell confocal microscopy. Preparation and mounting of the samples are key factors for successful live cell microscopy. To acquire biologically relevant data, it is necessary to minimize stress and avoid physical damage to plant tissues during the installation of the sample into the microscope. This is challenging, particularly when the whole plant is mounted as the living sample needs to be properly anchored in the microscopic system to obtain high-quality and high-resolution data. Here, we present a user-optimized sample holder EasyClick for live cell inverted confocal microscopic analysis of plant roots with diameters from 0.3 to 0.7 mm. The EasyClick holder was tested on an inverted confocal microscope using germinating plants of several cereals. Nevertheless, it can be directly used on other types of inverted microscopes from various producers and on different plant species. The EasyClick holder effectively restricts root lateral and vertical movements. This greatly improves the conditions for time-lapse microscopy of the samples of interest.

• Keywords
• Barley, Confocal microscopy, EasyClick, Growth, Hordeum vulgare, Live cell imaging, Plant, Root,
• MeSH
• Microscopy, Confocal MeSH
• Plant Roots * MeSH
• Publication type
• Journal Article MeSH

Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

• Keywords
• Ensifer meliloti, Alfalfa, MAPKs, SIMK, immunolocalization, infection pocket, infection thread, light-sheet fluorescence microscopy, root hairs, subcellular localization,
• MeSH
• Medicago sativa genetics   metabolism   MeSH
• Microscopy MeSH
• Mitogen-Activated Protein Kinases * metabolism   MeSH
• Plants metabolism   MeSH
• Sinorhizobium meliloti * metabolism   MeSH
• Symbiosis physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitogen-Activated Protein Kinases * MeSH

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

• Keywords
• Golgi, endocytosis, exocytosis, microscopy, proteomics, vesicle,
• MeSH
• Biological Transport MeSH
• Endocytosis MeSH
• Mass Spectrometry methods   MeSH
• Proteome * analysis   metabolism   MeSH
• Proteomics * methods   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Proteome * MeSH

The ability of plants to sense and orient their root growth towards gravity is studied in many laboratories. It is known that manual analysis of image data is subjected to human bias. Several semi-automated tools are available for analysing images from flatbed scanners, but there is no solution to automatically measure root bending angle over time for vertical-stage microscopy images. To address these problems, we developed ACORBA, which is an automated software that can measure root bending angle over time from vertical-stage microscope and flatbed scanner images. ACORBA also has a semi-automated mode for camera or stereomicroscope images. It represents a flexible approach based on both traditional image processing and deep machine learning segmentation to measure root angle progression over time. As the software is automated, it limits human interactions and is reproducible. ACORBA will support the plant biologist community by reducing labour and increasing reproducibility of image analysis of root gravitropism.

• Keywords
• UNET, deep machine learning, image segmentation, python, root gravitropism,
• Publication type
• Journal Article MeSH

Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.

• MeSH
• Arabidopsis genetics   growth & development   MeSH
• Cell Membrane genetics   metabolism   MeSH
• Genetic Variation MeSH
• Genotype MeSH
• Plant Roots genetics   growth & development   MeSH
• Mutation MeSH
• Organogenesis, Plant genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant MeSH
• Trichomes genetics   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Annexin 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins in plants. Generally, annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking, and stress responses. The expression of annexins is developmentally regulated, and it is sensitive to the external environment. ANN1 is expressed in almost all Arabidopsis tissues, while the most abundant is in the root, root hairs, and in the hypocotyl epidermal cells. Annexins were also occasionally proposed to associate with cytoskeleton and vesicles, but they were never developmentally localized at the subcellular level in diverse plant tissues and organs. Using advanced light-sheet fluorescence microscopy (LSFM), we followed the developmental and subcellular localization of GFP-tagged ANN1 in post-embryonic Arabidopsis organs. By contrast to conventional microscopy, LSFM allowed long-term imaging of ANN1-GFP in Arabidopsis plants at near-environmental conditions without affecting plant viability. We studied developmental regulation of ANN1-GFP expression and localization in growing Arabidopsis roots: strong accumulation was found in the root cap and epidermal cells (preferentially in elongating trichoblasts), but it was depleted in dividing cells localized in deeper layers of the root meristem. During root hair development, ANN1-GFP accumulated at the tips of emerging and growing root hairs, which was accompanied by decreased abundance in the trichoblasts. In aerial plant parts, ANN1-GFP was localized mainly in the cortical cytoplasm of trichomes and epidermal cells of hypocotyls, cotyledons, true leaves, and their petioles. At the subcellular level, ANN1-GFP was enriched at the plasma membrane (PM) and vesicles of non-dividing cells and in mitotic and cytokinetic microtubular arrays of dividing cells. Additionally, an independent immunolocalization method confirmed ANN1-GFP association with mitotic and cytokinetic microtubules (PPBs and phragmoplasts) in dividing cells of the lateral root cap. Lattice LSFM revealed subcellular accumulation of ANN1-GFP around the nuclear envelope of elongating trichoblasts. Massive relocation and accumulation of ANN1-GFP at the PM and in Hechtian strands and reticulum in plasmolyzed cells suggest a possible osmoprotective role of ANN1-GFP during plasmolysis/deplasmolysis cycle. This study shows complex developmental and subcellular localization patterns of ANN1 in living Arabidopsis plants.

• Keywords
• Arabidopsis thaliana, annexin 1, cell division, development, lattice light-sheet fluorescence microscopy, light-sheet fluorescence microscopy, plasma membrane, subcellular localization,
• Publication type
• Journal Article MeSH

Pattern formation, cell proliferation, and directional cell growth, are driving factors of plant organ shape, size, and overall vegetative development. The establishment of vegetative morphogenesis strongly depends on spatiotemporal control and synchronization of formative and proliferative cell division patterns. In this context, the progression of cell division and the regulation of cell division plane orientation are defined by molecular mechanisms converging to the proper positioning and temporal reorganization of microtubule arrays such as the preprophase microtubule band, the mitotic spindle and the cytokinetic phragmoplast. By focusing on the tractable example of primary root development and lateral root emergence in Arabidopsis thaliana, genetic studies have highlighted the importance of mechanisms underlying microtubule reorganization in the establishment of the root system. In this regard, severe alterations of root growth, and development found in extensively studied katanin1 mutants of A. thaliana (fra2, lue1, and ktn1-2), were previously attributed to defective rearrangements of cortical microtubules and aberrant cell division plane reorientation. How KATANIN1-mediated microtubule severing contributes to tissue patterning and organ morphogenesis, ultimately leading to anisotropy in microtubule organization is a trending topic under vigorous investigation. Here we addressed this issue during root development, using advanced light-sheet fluorescence microscopy (LSFM) and long-term imaging of ktn1-2 mutant expressing the GFP-TUA6 microtubule marker. This method allowed spatial and temporal monitoring of cell division patterns in growing roots. Analysis of acquired multidimensional data sets revealed the occurrence of ectopic cell divisions in various tissues including the calyptrogen and the protoxylem of the main root, as well as in lateral root primordia. Notably the ktn1-2 mutant exhibited excessive longitudinal cell divisions (parallel to the root axis) at ectopic positions. This suggested that changes in the cell division pattern and the occurrence of ectopic cell divisions contributed significantly to pleiotropic root phenotypes of ktn1-2 mutant. LSFM provided evidence that KATANIN1 is required for the spatiotemporal control of cell divisions and establishment of tissue patterns in living A. thaliana roots.

• Keywords
• Arabidopsis, ectopic cell division, katanin, light-sheet fluorescence microscopy, live cell imaging, microtubules, root development,
• Publication type
• Journal Article MeSH

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.

• Keywords
• A. thaliana, SPIM, cell biology, flower, germline, light sheet microscopy, live cell imaging, meiosis, plant biology,
• MeSH
• Arabidopsis cytology   growth & development   MeSH
• Cell Differentiation * MeSH
• Cytogenetic Analysis MeSH
• Flowers cytology   growth & development   MeSH
• Microscopy methods   MeSH
• Germ Cells, Plant cytology   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND AND AIMS: The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. METHODS: Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. KEY RESULTS: Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. CONCLUSIONS: Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern through deregulation of CDP orientation. The results suggest that the der1-3 mutation in the ACT2 gene does not influence solely root hair formation process, but also has more general effects on the actin cytoskeleton, plant growth and development.

• MeSH
• Actins genetics   metabolism   MeSH
• Arabidopsis genetics   growth & development   metabolism   MeSH
• Plant Roots growth & development   metabolism   MeSH
• Mutation * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACT2 protein, Arabidopsis MeSH Browser
• Actins MeSH
• Arabidopsis Proteins MeSH

Phospholipase D alpha 1 (PLDα1, At3g15730) and its product phosphatidic acid (PA) are involved in a variety of cellular and physiological processes, such as cytoskeletal remodeling, regulation of stomatal closure and opening, as well as biotic and abiotic stress signaling. Here we aimed to study developmental expression patterns and subcellular localization of PLDα1 in Arabidopsis using advanced microscopy methods such as light-sheet fluorescence microscopy (LSFM) and structured illumination microscopy (SIM). We complemented two knockout pldα1 mutants with a YFP-tagged PLDα1 expressed under the PLDα1 native promoter in order to study developmental expression pattern and subcellular localization of PLDα1 in Arabidopsis thaliana under natural conditions. Imaging of tissue-specific and developmentally-regulated localization of YFP-tagged PLDα1 by LSFM in roots of growing seedlings showed accumulation of PLDα1-YFP in the root cap and the rhizodermis. Expression of PLDα1-YFP in the rhizodermis was considerably higher in trichoblasts before and during root hair formation and growth. Thus, PLDα1-YFP accumulated in emerging root hairs and in the tips of growing root hairs. PLDα1-YFP showed cytoplasmic subcellular localization in root cap cells and in cells of the root transition zone. In aerial parts of plants PLDα1-YFP was also localized in the cytoplasm showing enhanced accumulation in the cortical cytoplasmic layer of epidermal non-dividing cells of hypocotyls, leaves, and leaf petioles. However, in dividing cells of root apical meristem and leaf petiole epidermis PLDα1-YFP was enriched in mitotic spindles and phragmoplasts, as revealed by co-visualization with microtubules. Finally, super-resolution SIM imaging revealed association of PLDα1-YFP with both microtubules and clathrin-coated vesicles (CCVs) and pits (CCPs). In conclusion, this study shows the developmentally-controlled expression and subcellular localization of PLDα1 in dividing and non-dividing Arabidopsis cells.

• Keywords
• Arabidopsis thaliana, At3g15730, development, light-sheet fluorescence microscopy, localization, microtubules, phospholipase D,
• Publication type
• Journal Article MeSH