Name: mycorrhizal maps as a tool to explore colonization patterns and fungal strategies in the roots of festuca rubra and zea mays
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Description: Watch this Scientific Journal Video about Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays at JoVE.com

This paper uses data resulting from two Ph.D. studies in the thematic area of &#34;Corn Mycorrhizal Patterns Driven by Agronomic Inputs&#34;, conducted by Victoria Pop-Moldovan, and &#34;Mycorrhizal Status and Development of Colonization in Mountain Grassland Dominant Species&#34;, conducted by Larisa Corcoz, under the coordination of Prof. Dr. Roxana Vidican.

1. Selection of biological material, root sampling, and storage
<ol>
	<li>Collect the entire root of plants with a shovel (Figure 1A) separately for each variant and replicate. Remove gently, by hand, the large soil aggregates from the roots. Wash the entire root system and measure it on a scale with 1 cm x 1 cm cells (Figure 1B). Cut the roots separately for each plant, and place them into a plastic bag.</li>
	<li>Collect all the clean roots ...

<ol>
	<li>Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-microbiome+interactions:+From+community+assembly+to+plant+health.">Plant-microbiome interactions: From community assembly to plant health.</a> Nature Reviews Microbiology. 18 (11), 607-621 (2020).</li><li>Jeffries, P., Barea, J. M., Hock, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4+Arbuscular+Mycorrhiza:+A+Key+Component+of+Sustainable+Plant-Soil+Ecosystems.">4 Arbuscular Mycorrhiza: A Key Component of Sustainable Plant-Soil Ecosystems.</a> The Mycota. IX Fungal Associations. , 51-75 (2012).</li><li>Parniske, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbuscular+mycorrhiza:+the+mother+of+plant+root+endosymbioses.">Arbuscular mycorrhiza: the mother of plant root endosymbioses.</a> Nature Reviews Microbiology. 6 (10), 763-775 (2008).</li><li>Gianinazzi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agroecology:+The+key+role+of+arbuscular+mycorrhizas+in+ecosystem+services.">Agroecology: The key role of arbuscular mycorrhizas in ecosystem services.</a> Mycorrhiza. 20 (8), 519-530 (2010).</li><li>Lee, E. -. H., Eo, J. -. K., Ka, K. -. H., Eom, A. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+arbuscular+mycorrhizal+fungi+and+their+roles+in+ecosystems.">Diversity of arbuscular mycorrhizal fungi and their roles in ecosystems.</a> Mycobiology. 41 (3), 121-125 (2013).</li><li>Zhang, Y., Zeng, M., Xiong, B., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+significance+of+arbuscular+mycorrhiza+biotechnology+in+modern+agricultural+system.">Ecological significance of arbuscular mycorrhiza biotechnology in modern agricultural system.</a> Ying Yong Sheng Tai Xue Bao = The Journal of Applied Ecology. 14 (4), 613-617 (2003).</li><li>Shah, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+endophytic+Bacillus+megaterium+colonization+on+structure+strengthening,+microbial+community,+chemical+composition+and+stabilization+properties+of+Hybrid+Pennisetum.">Effect of endophytic Bacillus megaterium colonization on structure strengthening, microbial community, chemical composition and stabilization properties of Hybrid Pennisetum.</a> Journal of the Science of Food and Agriculture. 100 (3), 1164-1173 (2020).</li><li>Souza, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Arbuscular Mycorrhizal Fungi. , (2015).</li><li>Sun, X. -. G., Tang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+routinely+used+methods+for+assessing+root+colonization+by+arbuscular+mycorrhizal+fungi.">Comparison of four routinely used methods for assessing root colonization by arbuscular mycorrhizal fungi.</a> Botany. 90 (11), 1073-1083 (2012).</li><li>Smith, S., Read, D., Smith, S. E., Read, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+Roots+and+Anatomy+of+Arbuscular+Mycorrhiza.">Colonization of Roots and Anatomy of Arbuscular Mycorrhiza.</a> Mycorrhizal Symbiosis. , 42-90 (2008).</li><li>Stoian, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+approach+and+future+perspectives+in+microscopic+patterns+of+mycorrhizal+roots.">Sensitive approach and future perspectives in microscopic patterns of mycorrhizal roots.</a> Scientific Reports. 9 (1), 10233 (2019).</li><li>Pop-Moldovan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergence+in+corn+mycorrhizal+colonization+patterns+due+to+organic+treatment.">Divergence in corn mycorrhizal colonization patterns due to organic treatment.</a> Plants. 10 (12), 2760 (2021).</li><li>Corcoz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycorrhizal+patterns+in+the+roots+of+dominant+Festuca+rubra+in+a+high-natural-value+grassland.">Mycorrhizal patterns in the roots of dominant Festuca rubra in a high-natural-value grassland.</a> Plants. 11 (1), 112 (2021).</li><li>Corcoz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+colonization+strategies+in+roots+of+long-term+fertilized+Festuca+rubra.">Deciphering the colonization strategies in roots of long-term fertilized Festuca rubra.</a> Agronomy. 12 (3), 650 (2022).</li><li>Stoian, V., Florian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycorrhiza+-+Benefits,+influence,+diagnostic+method.">Mycorrhiza - Benefits, influence, diagnostic method.</a> Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Agriculture. 66 (1), 2009 (2009).</li><li>Prates J&#250;nior, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agroecological+coffee+management+increases+arbuscular+mycorrhizal+fungi+diversity.">Agroecological coffee management increases arbuscular mycorrhizal fungi diversity.</a> PLoS One. 14 (1), 0209093 (2019).</li><li>Rillig, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+farmers+should+manage+the+arbuscular+mycorrhizal+symbiosis.">Why farmers should manage the arbuscular mycorrhizal symbiosis.</a> New Phytologist. 222 (3), 1171-1175 (2019).</li><li>Rillig, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+integrated+mycorrhizal+technology:+Harnessing+mycorrhiza+for+sustainable+intensification+in+agriculture.">Towards an integrated mycorrhizal technology: Harnessing mycorrhiza for sustainable intensification in agriculture.</a> Frontiers in Plant Science. 7, 1625 (2016).</li><li>Bhale, U. N., Bansode, S. A., Singh, S., Gehlot, P., Singh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifactorial+Role+of+Arbuscular+Mycorrhizae+in+Agroecosystem.">Multifactorial Role of Arbuscular Mycorrhizae in Agroecosystem.</a> Fungi and their Role in Sustainable Development: Current Perspectives. , 205-220 (2018).</li><li>Khaliq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+control+of+dry+root+rot+of+chickpea+caused+by+Rhizoctonia+bataticola+under+the+natural+field+condition.">Integrated control of dry root rot of chickpea caused by Rhizoctonia bataticola under the natural field condition.</a> Biotechnology Reports. 25, 00423 (2020).</li><li>Vaida, I., P&#259;curar, F., Rotar, I., Tomo&#537;, L., Stoian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+diversity+due+to+long-term+management+in+a+high+natural+value+grassland.">Changes in diversity due to long-term management in a high natural value grassland.</a> Plants. 10 (4), 739 (2021).</li><li>. The International Collection of (Vesicular) Arbuscular Mycorrhizal Fungi Available from: <a target="_blank" href="https://invam.wvu.edu/collection">https://invam.wvu.edu/collection</a> (2022)</li><li>. The International Bank for the Glomeromycota Available from: <a target="_blank" href="https://www.i-beg.eu/">https://www.i-beg.eu/</a> (2022)</li></ol>

Studies on mycorrhizal colonization are vital for new strategy development in the agronomic field. The potential of multiple cultivated plants to form a symbiotic association with arbuscular mycorrhizas made them an important component of the agroecosystem&#39;s sustainable development and the maintenance of its health16,17,18,19,20. Thus, there is a need for ...

Arbuscular mycorrhiza (AM) fungi are a category of soil-borne endophytes that are constantly an area of interest for researchers. Their presence in the roots of most plants and their involvement in nutrient cycles makes them vital components in the stability of every ecosystem where herbaceous plants are present1,2. Through their extra-radicular mycelium, AM act as a fungal extension for plant roots, especially in hard-to-reach areas3. The main activity is in host plant roots, where AM develops large hyphae networks and specific intracellular structures called arbuscules. The lack of host specificity allows the symbiont to colonize multiple species at the same time. This ability provides AM with the role of resource allocation and nutrient regulation in the ecosystem; the fungus also provides support in plant survival and aids in plant performance4,5,6,7. The reaction of AM species to host roots is visible in the extension and location of the intra-radicular mycelium and the presence and shape of the arbuscules developed intracellularly. The intracellular arbuscules act as an interchange point between the two symbionts and represent areas characterized by fast transfer processes. The structures that the AM produce are species-dependent, and, in addition to arbuscules, in the roots, they also develop vesicles, spores, and auxiliary cells.
There are many challenges in the assessment of AM symbionts in plant roots8,9. The first one is their constant development during the entire vegetation period of hosts, which leads to multiple changes in the hyphal arbuscular structure. The different stages of arbuscular growth, up to their collapse, are clearly present in roots, but the senescent AM structures are sometimes digested, which makes them only partially visible10. The second challenge is represented by the staining method and protocol, the large diversity of root systems, the dimension of their cells, and the differences in thickness, which make it hard to propose a unified method. The last challenge is represented by the assessment and scoring of AM colonization. There are numerous methods that score AM with different degrees of objectivity, and most of them are still restricted to microscopy techniques. The simple ones are based on the presence/absence of structures in the root cortex, while the more complex ones are based on visual scoring and the use of colonization classes, with the integration of the frequency and intensity of the colonization phenomenon. A lot of data have been produced in the last decades on the mycorrhizal status of multiple species, but most of the methods are restricted to the observed value of colonization without pointing to the real position of each structure in the root cortex. As a response to the necessity of more accurate results on AM colonization, a method based on microscopic analysis of mycorrhizal patterns (MycoPatt) in roots was developed to assemble, in a digital form, the detailed mycorrhizal maps11. Also, the method allows the objective calculation of colonization parameters and the determination of the actual position of each structure in the root.
The position of the AM fungal structures can be important in answering the following two questions. The first one is related to the analysis of the colonization in one specific moment from the vegetation cycle of a plant. In this context, it is very useful to observe the arbuscular/vesicle abundance, report how are they located in the root, and provide a very clear colonization image and parameters. The second one is related to the detection of fungal strategy and its orientation and even the forecast of its future development. One application of the MycoPatt can be for plants analyzed daily, every 2-3 days, weekly, or during various growth stages. In this context, the location of the vesicles/arbuscules is important to better understand the biological mechanism of AM colonization. These parameters and observations are very useful to supplement the mathematical parameters.
The aim of this article is to demonstrate the ability of the MycoPatt system to explore the native AM fungi colonization potential and strategy in Zea mays (corn) roots during different development stages and in Festuca rubra (red fescue) roots under different long-term fertilization conditions. To fulfill the aim, two large databases from two experiments were analyzed. The corn experiment was established at Cojocna (46&#176;44&#8242;56&#8243; lat. N and 23&#176;50&#8242;0&#8243; long. E), in the Experimental Didactic Farm of the University of Agricultural Sciences and Veterinary Medicine Cluj on a phaeoziom with a loamy texture soil12. The red fescue experiment is a part of a larger experimental site established in 2001 in Ghe&#539;ari, Apuseni Mountains (46&#176;49&#39;064&#34; lat. N and 22&#176;81&#39;418&#39;&#39; long. E), on a preluvosol (terra rossa) soil type13,14. Corn was collected in five different growth phenophases12: B1 = 2-4 leaves (as a control point for the start of mycorrhizal colonization); B2 = 6 leaves; B3 = 8-10 leaves; B4 = cob formation; B5 = physiological maturity. Starting from the 2-4 leaves stage (A0), an organic treatment was applied, which resulted in a two-graduation factor (A1 = control and A2 = treated). Roots of red fescue were collected at flowering from an experiment with five long-term fertilizations13,14: V1 = control, non-fertilized; V2 = 10 t&#183;ha-1 manure; V3 = 10 t&#183;ha-1 manure + N&#160;50 kg&#183;ha-1, P2O5 25 kg&#183;ha-1, K2O 25 kg&#183;ha-1; V4 = N 100 kg&#183;ha-1, P2O5 50 kg&#183;ha-1, K2O 50 kg&#183;ha-1; V5 = 10 t&#183;ha-1 manure + N 100 kg&#183;ha-1, P2O5 50 kg&#183;ha-1, K2O 50 kg&#183;ha-1. Five plants were collected in each development stage from every fertilization variant. The staining protocols and their performance in terms of sample processing time and quality of staining were analyzed. The relation between AM hyphae development and the presence of its structures in roots was analyzed separately for each species and continued with the identification of the most permissive roots for colonization. The specific colonization patterns of each root system were analyzed based on colonization maps and the value of AM parameters.
Corn is an annual plant, which implies continuous growth of the roots, and that was the main reason to apply the MycoPatt in the growing stages. Red fescue is a perennial plant from a grassland treated for a long time with different fertilizers. Its roots have a shorter development of 1 year, and the anthesis is considered as the vegetation point when the plant changes its metabolism from vegetative to generative. To catch these plants during these intense activity periods, the abovementioned time points were chosen. Sampling during the vegetation period is difficult for this species when grown in natural grasslands.

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mycorrhizal maps as a tool to explore colonization patterns and fungal strategies in the roots of festuca rubra and zea mays

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in the ecosystems. The colonization process is dependent on several factors like soil ecology, the genetic diversity of the fungi and host, and agronomic practices. Their synchronized action leads to the development of a complex hyphal network and leads to the secondary development of vesicles and arbuscules in the root cells. The aim of this research was to analyze the efficiency of the mycorrhizal patterns (MycoPatt) method for the positioning of fungal structures in the roots of Festuca rubra and Zea mays. Another objective was to explore the fungal colonization strategy as revealed by mycorrhizal maps of each species. The acquisition and assemblage of multiple microscopic images allow mycorrhizal colonization assessment in both corn and red fescue plants to provide information on the realistic position of the developed structures. The observed mycorrhizal patterns highlight the variable efficiency of each plant in terms of developing connections with soil symbiotic fungi, caused by applied treatments and growth stage. Mycorrhizal detailed maps obtained through the MycoPatt method are useful for the early detection of plant efficiency in symbiotic acquisition from the soil.

The correct use of the gentle crushing method of the roots after the staining procedures provides good details of mycorrhizal structures, both for Zea mays (Figure 8A-C) and Festuca rubra (Figure 9A-E), good contrast between mycorrhizal structures and root cells, and a confirmation of the stele due to the blue color. If the clearing and staining procedures fail to succeed, root...

Watch this Scientific Journal Video about Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays at JoVE.com

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

The protocol here describes the methods for the assessment of the arbuscular mycorrhizal colonization patterns and strategy in roots for two species: Zea mays and Festuca rubra. The use of the MycoPatt method permits the calculation of parameters, the conversion of mycorrhizal structures into digital data, and the mapping of their real position in roots.

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

Arbuscular mycorrhizal fungi are symbionts in the roots of plants. Their role is to sustain host development and maintain the nutritional equilibrium in ...

MycoPatt method permits an in depth exploration of arbuscular mycorrhizal colonization in roots. Mycorrhizal maps point to fungal expansion and present the real position of each structure as a specific pattern. Mycorrhizal colonization is objectively analyzed at 1%resolution.Each root segment is automatically converted to a digital matrix and generates an extensive database of colonization parameters. To begin, remove the roots from the refrigerator and place them on a working platform for thawing at room temperature. To perform root clearing, place all roots from one plant in a 30 to 50 milliliter jar, then prepare a 10%sodium hydroxide solution with tap water and pour it into the jar until it completely covers the roots.Shake the jar vigorously for one to two minutes for homogenous dispersion of clearing solution in the roots. Allow the roots to remain in the clearing solution for 48 hours. For root rinsing, pass the content of the jar through a sieve.Rinse the roots several times in tap water until the clearing solution is completely removed. Next place the rinsed roots in a clean jar for staining. Then prepare a five to five percent ink vinegar solution with tap water.Pour it into the jar until it covers the roots and shake it for one to two minutes. After 48 hours for partial destaining, rinse the stained roots in tap water for one to two minutes. Shake the jar vigorously to remove the extra staining solution.Repeat the procedure until the roots are clear for microscopic evaluation. For root segmentation, place the stained roots on a scaled cutting board. Cut the roots into one centimeter segments.Choose 15 segments for each variant. Next, spread the roots on a slide for segment preparation, use a laminating pouch to cover the roots and gently crush them, starting from an edge. Use a soft plastic tool to display the roots on the slide.Carefully remove the laminating pouch and cover the sample with a cover slip. Add water to a corner of the slide with a pipette, and let the water slowly spread on the slide. Remove the extra water with a paper towel.For microscopic analysis, use a microscope equipped with a good resolution camera. Analyze the slides starting from an extremity. Capture each microscopic field.Use 10 or 40x magnification for thick roots and 40x for thin roots. After capturing, rename each image with a code that will permit the real post-assemblage of root parts. Use presentation software to design a drawing board for image assemblage, set the width two to three centimeters wider than the image width.After capturing 15 pictures, add all captured images from one segment in the order of their capture, starting with one to 15, and reconstruct the entire length of the root segment. Next, use the vertical alignment to align the images in the center and ensure that each image follows the previous one. Place a grid of 10 by 150 cells on all pictures to cover the entire root segment.Additionally, place a 10 by 10 grid on each picture and in every cell of this grid, insert a number from one to six if an arbuscular mycorrhizal or AM structure is visible, or leave blank if no AM structure is present. Add a table for a grid of 10 cells in width and 150 cells in length. Change the table with dimensions to the width of the images, then change the length of the table to comprise all the images.For scoring mycorrhizal colonization, use the unique number to score each type of structure as described in the mycorrhizal pattern method. Use one for hyphae, two for arbuscules, three for vesicles, four for spores, five for auxiliary cells, and six for entry points. Score each observed mycorrhizal structure from each cell of the previously applied grids.After scoring, insert all the obtained scores into the MycoPatt spreadsheet. Use the copy paste function to transfer all the scores from the presentation into the first sheet named Raw Data. For the primary analysis, use the third sheet named Parameters to visualize the results as percentages separately in three forms.Use columns A to K to analyze the horizontal image of colonization, columns M to W for the vertical image of colonization, and columns Y to AI to analyze the transversal colonization for each of the 15 10 by 10 squares and the final average colonization. Next, apply the definitions and formulas specific to each parameter. For production and extraction of mycorrhizal maps, visualize the image obtained from the conversion of mycorrhizal structures code into colors in the second sheet named Graphs.Then export the resultant image in the graph sheet as an image and use the color code in the legend to analyze mycorrhizal patterns. For mycorrhizal map analysis, identify the most important structures and their assemblage. Then describe the mycorrhizal colonization pattern observed in the analyzed roots, followed by a colonization strategy based on observed structural development in the root, the branching pattern, and arbuscular vesicle development.This protocol provides the details of mycorrhizal structures with good contrast between mycorrhizal structures and root cells for Zea mays and Festal rubra. The unsuccessful clearing and staining procedures resulted in unclear microscopic images of mycorrhizal structures in Festal rubra and Zea mays. Mycorrhizal colonization patterns allowed a complete exploration of the colonization mechanism, this method provides a deep, small scale exploration of colonization patterns and strategies for Zea mays and Festuca rubra with an additional visual expression of colonization parameters.In Zea mays highly fluctuating colonization potential depending on the plant's developmental stage was observed, however, in Festuca rubra, the colonization processes occur inside the roots and the development of hyphal networks was correlated with a low development speed of the roots. MycoPatt is suitable for mapping the arbuscular mycorrhizal colonization in any plant. It is also helpful to fully explore the colonization mechanism and assess fungal strategy along the roots.

mycorrhizal-maps-as-tool-to-explore-colonization-patterns-fungal

Research

JoVE Journal

Biology

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.