Name: shootward movement of cfda tracer loaded in the bottom sink tissues of arabidopsis
Uploaded: 2019-05-11T15:00:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis at JoVE.com

This work was funded by National Natural Science Foundation of China (31671257) and Hubei Collaborative Innovation Center for Grain Industry (LXT-16-18).

1. Arabidopsis vertical growth in MS medium
<ol>
	<li>The interior of laminar flow cabinet needs to be treated with 30 min UV light and 15 min airflow before usage. Make sure to close the glass door when UV light is on. Spray all tools and plates with 70% ethanol before placing them in the cabinet.</li>
	<li>Prepare the Murashige and Skoog (MS) medium in a standard 90 mm-diameter Petri dish under a laminar flow cabinet. 
	NOTE: The MS medium contains the following components: 20.6 mM NH4NO3, 18.8 mM KNO3, 1.25 mM KH2PO4, 1.5 mM MgSO4&#183;7H2O, 3 mM CaCl2&#183;2H2O, 0.1 mM MnSO4&#183;H2O, 1.03 &#181;M Na2MoO4&#183;2H2O, 0.1 mM H3BO3, 30 &#181;M ZnSO4&#183;7H2O, 0.1 &#181;M CuSO4&#183;5H2O, 0.1 &#181;M CoCl2&#183;6H2O, 1.39 &#181;M KI, 97 &#181;M FeSO4&#183;7H2O, 114.5 &#181;M ethylenediaminetetraacetic acid (EDTA), 4.07 &#181;M nicotinic Acid, 2.44 &#181;M pyridoxine HCl, 0.15 &#181;M thiamine HCl, 2.68 mM glycine, 555 &#181;M myo-inositol, 87.7 mM sucrose and 10 g/L agar.</li>
	<li>Moisturize the sterilized tips or toothpicks by touching the tip or toothpicks to the MS medium, then dip the sterilized seeds one by one onto the fixed position of each Petri dish indicated by the seeding card.</li>
	<li>Seal the Petri dish with paraffin film and sticky tape and place it vertically on a clear stand in a growth room (22 &#176;C, 70% moisture) with a day cycle of 16 h of light and 8 h of darkness (lighting from 6 am to 10 pm). The Arabidopsis plant is ready for CFDA loading on day 9-13 after sowing. 
	NOTE: The following procedure is performed in the afternoon from 2 pm to 5 pm.</li>
</ol>
2. CFDA loading with the root cutting procedure
<ol>
	<li>Prepare fresh CFDA working solution immediately before use. Dilute the 1 mM CFDA stock solution stored in -20 &#176;C freezer with sterile ultrapure water to a working concentration of 5 &#181;M.</li>
	<li>Cut the micro-porous paraffin membrane film (see Table of Materials) into small pieces of 3 mm x 3 mm size.</li>
	<li>Uncover the growing plants in the room at 22 &#176;C and clear the excess moisture with a paper towel. Place the small film pieces below each root. 
	NOTE: From this to step 2.6, the whole process should be completed within 15 min.</li>
	<li>Lift the plants onto the Petri dish lid. Cut the roots in a position about 5-10 mm below the root-hypocotyl junction. Place the shoot part back on to the film on the medium.</li>
	<li>Carefully apply 0.25 &#181;L of CFDA onto the cutting end of each root under a dissecting microscope. Avoid splashing to other parts of plant.</li>
	<li>Cover the plate and leave the plants under light for 2-3 h (22 &#176;C) in the growth room.</li>
	<li>Rinse the stained plants three times sequentially in three separate beakers filled with distilled water, then observe the plants under a stereo-fluorescence microscope with zoom from 1.4x to 3.3x (see the Table of Materials). For fluorescence detection, use a high efficiency filter cube (470/20 nm excitation, 495 nm split and 525/50 nm emission) mounted to transmit the fluorescence signal.</li>
</ol>
3. CFDA loading with hypocotyl-pinching procedure
<ol>
	<li>Prepare fresh CFDA working solution immediately before use. Dilute the 1 mM CFDA stock solution stored in -20 &#176;C freezer with sterile ultrapure water (see the Table of Materials) to a working concentration of 5 &#181;M.</li>
	<li>Cut the micro-porous paraffin membrane film (see Table of Materials) into small pieces of 3 mm x 3 mm size.</li>
	<li>Uncover the growing plants in the room at 22 &#176;C and clear the excess moisture with a paper towel. 
	NOTE: From this to step 3.7, the whole process should be completed within 15 min.</li>
	<li>Lay a small piece of film under the root-hypocotyl junction of each plant.</li>
	<li>Use forceps to gently pinch the hypocotyl near to the root-hypocotyl junction under a dissecting microscope.</li>
	<li>Carefully apply 0.1 &#181;L of CFDA onto the wound site under a dissecting microscope. Avoid splashing to other parts of plant.</li>
	<li>Cover the plate, and leave the plants under light (22 &#176;C) for 2-3 h.</li>
	<li>Rinse the stained plants three times sequentially in three separate beakers filled with distilled water, then observe the plants under a stereo-fluorescence microscope with zoom from 1.4x to 3.3x (see the Table of Materials). For fluorescence detection, mount a high efficiency filter cube (470/20 nm excitation, 495 nm split and 525/50 nm emission) to transmit the fluorescence signal.</li>
</ol>

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The authors have nothing to disclose.

Emerging studies have shown that plants can rapidly respond to external stimuli23, including manipulation introduced to the experimental procedures22. In our initial experiment, our oversight of this knowledge often leads to staining failure. With these lessons, we suggest that the following precautions should be kept in mind when performing this experiment: (1) the seeds after harvest should be kept in a storage cabinet set to a low temperature and low moisture; (2) manipu...

Many fluorescent tracers with a range of spectral properties, such as 5(6)-carboxyfluorescein (CF)1, 8-hydroxypyrene-1,3,6-trisulphonic acid2, Lucifer yellow CH (LYCH)3, Esculin and CTER4, have been developed and applied in plants to monitor symplastic movement and phloem activity. Generally, a symplastic dye is loaded into a cut in the target tissue and the sequential dispersion of the reporter into other parts of plant will demonstrate the intercellular communication. Although the mechanism of dye absorption is not fully understood, the principle underlying CF movement inside live cells has been widely acknowledged. The ester form of CF (CF diacetate, CFDA) is non-fluorescent, but membrane-permeable. This property allows rapid membrane diffusion of the dye into cells. Once inside live cells, intracellular esterases remove the acetate groups at the 3&#39; and 6&#39; position of CFDA, releasing the fluorescent and membrane-impermeable CF (Figure 1, alternatively refer Wright et al.2); CF can then move through the plasmodesmata to other parts of plants.
A well-established procedure with CFDA is that it can be loaded into source leaves and used to monitor the phloem streaming and phloem unloading in the sink tissues of many species, e.g., as CF unloading in the Arabidopsis root5, phloem unloading during potato tuberization6, phloem unloading in the Nicotiana sink leaves7, and so on. By similar loading approaches, other studies have adopted this dye to demonstrate the symplastic connection between host and parasite8,9, or to reveal the symbiotic relationships10,11.
Another way to make use of this dye is to load it into specific cells or single cell by microinjection to determine its distribution pattern. Such sophisticated techniques have greatly facilitated our deeper understanding of plasmodesmata-mediated intercellular communication, particularly in the development of the concept of symplastic domain12,13. For example, the microinjection of CFDA into cotyledon cells of Arabidopsis resulted in the dye-coupling pattern in the hypocotyl epidermis but non-coupling in the underlying cells or in the root epidermis, therefore the hypocotyl epidermis forms a symplastic domain14. Similar domains, such as the stomatal guard cells15, sieve element-companion cells16, root hair cells14 and root cap17,18 have been identified by microinjection technique. Most surprisingly, some domains allow tracer molecules to move in a certain direction. Take the trichome domain for example, microinjection of a fluorescent probe into the supporting epidermal cell leads to the flow of tracer into the trichome domain, however, the reverse injection does not hold true19. A recent report has also found similar situations in the symplastic domains of the Sedum embryo20. Thus, all above cases imply that swapping of loading sites may lead to novel insights into symplastic communication. Our previous experiment aiming to dissect the route of root-to-shoot mobile silencing identified a novel symplastic domain, or the HEJ (Hypocotyl-epicotyl junction) zone, which was further verified through the root-loading (non-canonical sink-loading) CFDA experiment21. Here, we further elaborate the root-to-shoot CF movement by using a simple method and recover a potential symplastic domain by shifting the loading sites. Furthermore, this procedure may be adapted to differentiate genetic backgrounds that have altered root-to-shoot long-distance transport.

<table><tbody><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;&amp;nbsp;</td><td>Sinopharm Chemical Reagent</td><td>10017218</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sinopharm Chemical Reagent</td><td>10017608</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>10013018</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>20011160</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sinopharm Chemical Reagent</td><td>10013418</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>10019818</td><td></td></tr><tr><td>Boric Acid</td><td>Sinopharm Chemical Reagent</td><td>10004818</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>10024018</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>10008218</td><td></td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>10007216</td><td></td></tr><tr><td>KI</td><td>Sinopharm Chemical Reagent</td><td>10017160</td><td></td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent</td><td>10012118</td><td></td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sinopharm Chemical Reagent</td><td>10009717</td><td></td></tr><tr><td>NaOH</td><td>Sinopharm Chemical Reagent</td><td>10019718</td><td></td></tr><tr><td>KOH</td><td>Sinopharm Chemical Reagent</td><td>10017018</td><td></td></tr><tr><td>Sucrose</td><td>Sinopharm Chemical Reagent</td><td>10021418</td><td></td></tr><tr><td>Myo-inositol&amp;nbsp;&amp;nbsp;</td><td>MACKLIN</td><td>I811835</td><td></td></tr><tr><td>Nicotinic Acid&amp;nbsp;</td><td>MACKLIN</td><td>N814565</td><td></td></tr><tr><td>Pyridoxine HCl</td><td>MACKLIN</td><td>V820447</td><td></td></tr><tr><td>Thiamine HCl</td><td>MACKLIN</td><td>T818865</td><td></td></tr><tr><td>Glycine</td><td>MACKLIN</td><td>G800880</td><td></td></tr><tr><td>Agar powder</td><td>Novon</td><td>ZZ14022</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Zeiss</td><td>Axio Zoom V16</td><td></td></tr><tr><td>Dissecting microscope</td><td>SDPTOP</td><td>SRE-1030</td><td></td></tr><tr><td>200&amp;mu;l pipette</td><td>Dragon Laboratory Instruments</td><td>713111110000-20-200ul</td><td></td></tr><tr><td>2.5&amp;mu;l pipette</td><td>Eppendorf</td><td>3120000011</td><td></td></tr><tr><td>Fine forceps&amp;nbsp;</td><td>TWEEZERS</td><td>ST-15</td><td></td></tr><tr><td>Parafilm</td><td>PARAFILM</td><td>PM-996</td><td></td></tr><tr><td>Stainless steel double-sided blade</td><td>Gillette</td><td>Platinum-Plus Double-Edge Blade</td><td></td></tr></tbody></table>

shootward movement of cfda tracer loaded in the bottom sink tissues of arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Symplastic movement is often subject to environmental fluctuations. Perturbation of the plant growing state, and even the process of tissue preparation will affect the size exclusion limit of plasmodesmata, thus affecting the symplastic transport22. To improve the staining efficiency, we confine our operation in the growth room, where the temperature and moisture is tightly controlled, and also perform the whole procedure as quickly as possible (ideally within 10-1...

Watch this Scientific Journal Video about Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis at JoVE.com

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

shootward-movement-cfda-tracer-loaded-bottom-sink-tissues

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6.7K Views.  Yangtze University. The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Video: Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about chloroplast movement by studying the chloroplast positioning in leaves using e.g., confocal fluorescence microscopy, but access to this type of microscopy is limited. This protocol describes a method that uses the changes in leaf transmission as a proxy for chloroplast movement. If chloroplasts are spread out in order to maximize light interception, the transmission will be low. If chloroplasts move towards the anticlinal cell walls to avoid light, the transmission will be higher. This protocol describes how to use a straightforward, home-built instrument to expose leaves to different blue light intensities and quantify the dynamic changes in leaf transmission. This approach allows researchers to quantitatively describe chloroplast movement in different species and mutants, study the effects of chemicals and environmental factors on it, or screen for novel mutants e.g., to identify missing components in the process that leads from light perception to the movement of chloroplasts.

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Many plant species change the positioning of chloroplasts to optimize light absorption. This protocol describes how to use a straightforward, home-built instrument to investigate chloroplast movement in Arabidopsis thaliana leaves using changes in the transmission of light through a leaf as a proxy.

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Biology

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

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Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Fluorescence Activated Cell Sorting of Plant Protoplasts

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System