Name: an induction system for clustered stomata by sugar solution immersion treatment in arabidopsis thaliana seedlings
Uploaded: 2019-02-15T14:30:00
Description: Watch this Scientific Journal Video about An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings at JoVE.com

We are grateful to Prof. Seiichiro Hasezawa for his kind support of our work. This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) KAKENHgrant numbers 17K19380 and 18H05492, from The Sumitomo Foundation for a Grant for Basic Science Research Projects grant number 160146, and The Canon Foundation to T.H. This experimental system was developed under a financial support from the JSPS KAKENHgrant number 26891006 to K. A. We thank Robbie Lewis, MSc, from Edanz Group (www.edanzediting.com/ac) for editing a draft of the manuscript.

1. Preparation of 3% Sucrose-containing 1/2 Murashige-Skoog Medium Solution
<ol>
	<li>Add 1.1 g of Murashige-Skoog medium salts and 15 g of sucrose to a beaker.</li>
	<li>Add 490 mL of distilled water and mix well using a stir bar.</li>
	<li>Adjust the pH to 5.8 using KOH.</li>
	<li>Dilute to 500 mL with distilled water and transfer the solution into a medium bottle.</li>
	<li>Sterilize the solution by autoclaving (121 &#176;C, 20 min). If not used immediately, this solution may be kept at 4 &#176;C after sterilization...

<ol>
	<li>Raschke, K., Fellows, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+movement+in+Zea+mays:+shuttle+of+potassium+and+chloride+between+guard+cells+and+subsidiary+cells.">Stomatal movement in Zea mays: shuttle of potassium and chloride between guard cells and subsidiary cells.</a> Planta. 101 (4), 296-316 (1971).</li><li>Higaki, T., Hashimoto-Sugimoto, M., Akita, K., Iba, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+environmental+responses+of+PATROL1+in+Arabidopsis+subsidiary+cells.">Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells.</a> Plant and Cell Physiology. 55 (4), 773-780 (2013).</li><li>Bergmann, D. C., Sack, F. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+formation+of+chloroplast+protrusions+and+stromules+in+mesophyll+cells+of+Arabidopsis+thaliana.">Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana.</a> Protoplasma. 230 (1-2), 23-30 (2007).</li><li>Abe, T., Hashimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+microtubule+dynamics+by+expression+of+modified+&#945;-tubulin+protein+causes+right-handed+helical+growth+in+transgenic+Arabidopsis+plants.">Altered microtubule dynamics by expression of modified &#945;-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants.</a> The Plant Journal. 43 (2), 191-204 (2005).</li><li>Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+plant+cell+surface+dynamics+with+variable-angle+epifluorescence+microscopy.">Real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy.</a> Journal of Visualized Experiments. 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We have presented protocols for induction of clustered stomata in A. thaliana seedlings by immersion treatment with a sucrose-containing medium solution. As shown here, this method is very simple and requires no specialized skill but can efficiently induce clustered stomata. More than 45% of guard cells are clustered with 3% sucrose-containing medium solution (mean values of more than 20 independent observations)6. Moreover, this experimental system can be directly applied to transgenic o...

The plant stoma is an essential organ for gas exchange for photosynthesis and transpiration, and stomatal movement is accomplished by significant changes in guard cells through ion-driven uptake and release of water. Under a microscope, we can observe a spaced distribution pattern of stomata on the surfaces of leaves and stems. This spaced distribution of stomata is considered to help stomatal movement, which is regulated by ion and water exchange between guard cells and neighboring epidermal cells1,2. Experimental induction systems for clustered stomata are useful for investigating the importance of the spaced distribution of stomata.
It has been reported that spatial clustering of stomata can be induced by genetic modification of key genes for guard cell differentiation3,4 or treatment with a chemical compound5. We also reported that immersion treatment with a medium solution supplemented with sugars including sucrose, glucose, and fructose caused stomatal clustering in cotyledons of Arabidopsis thaliana seedlings6. Reduced callose in new cell walls separating meristemoids and epidermal cells was observed in the sucrose-treated cotyledon epidermis, suggesting that sucrose solution immersion treatment negatively affects the cell wall, which prevents the leakage and ectopic action of key gene products for guard cell differentiation (e.g. transcription factors) towards adjacent epidermal cells6. A similar mechanism was suggested from studies on gsl8/chor mutants7,8. Our experimental system for reproducible induction of clustered stomata using sucrose-containing medium solution is quite easy and cheap. It can also be used to investigate intracellular structures such as organelles and the cytoskeleton in the clustered guard cells when applied to transgenic lines expressing fluorescent markers that label intracellular structures9,10.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">24-well plate</td>
			<td style="width:135px;">Sumitomo Bakelite</td>
			<td style="width:168px;">MS-0824R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">488 nm laser</td>
			<td style="width:135px;">Furukawa Denko</td>
			<td style="width:168px;">HPU-50101-PFS2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">488 nm laser</td>
			<td style="width:135px;">Olympus</td>
			<td>Sapphire488-20/O</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">510 nm long-pass filter</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">BA510IF</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">524 - 546 nm band-pass filter</td>
			<td style="width:135px;">Semrock</td>
			<td style="width:168px;">FF01-535/22-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">530 nm short-pass filter</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">BA530RIF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">561 nm laser</td>
			<td style="width:135px;">CVI Melles Griot</td>
			<td style="width:168px;">85-YCA-025-040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">604 - 644 nm band-pass filter</td>
			<td style="width:135px;">Semrock</td>
			<td style="width:168px;">FF01-624/40-25</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Confocal laser scanning head</td>
			<td style="width:135px;">Yokogawa</td>
			<td style="width:168px;">CSU10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Confocal laser scanning head</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">FV300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cooled CCD camera</td>
			<td style="width:135px;">Photometrics</td>
			<td style="width:168px;">CoolSNAP HQ2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Image acquisition software</td>
			<td style="width:135px;">Molecular Devices</td>
			<td style="width:168px;">MetaMorph version 7.8.2.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Image acquisition software</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">FLUOVIEW v5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Immersion oil</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">Immersion Oil Type-F</td>
			<td>ne = 1.518 (23 degrees)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Inverted microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">IX-70</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Inverted microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">IX-71</td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:216px;">Murashige and Skoog Plant Salt Mixture</td>
			<td style="width:135px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:168px;">392-00591</td>
			<td style="width:167px;">Murashige T and Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3), 473-497.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective lens&#160;</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">UPlanApo 100x / 1.35 NA Oil Iris 1.35</td>
			<td>NA = 1.35</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective lens&#160;</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">UPlanAPO 40x / 0.85 NA</td>
			<td>NA = 0.85</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sucrose</td>
			<td style="width:135px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:168px;">196-00015</td>
			<td></td>
		</tr>
	</tbody>
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an induction system for clustered stomata by sugar solution immersion treatment in arabidopsis thaliana seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

Here, the protocol for a simple method of inducing stomatal clustering with sucrose-containing medium solution in A. thaliana seedlings has been presented. The clustered guard cells grown in sucrose-containing medium solution (Figure 1B) have larger chloroplasts than guard cells grown in sucrose-free control conditions (Figure 1A). The enlargement of chloroplasts was confirmed with CT-GF...

Watch this Scientific Journal Video about An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings at JoVE.com

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

This method have answered a key question of plant developmental biology. That is, what is the importance of spatial distribution of stomata? Our induction system for clustered stomata using sucrose solution is fairly easy and directly applicable to transgenic or mutant lines of Arabidopsis thaliana.To begin, add 1.1 grams of Murashige-Skoog medium salts and 15 grams of sucrose to a beaker. Add 490 milliliters of distilled water to the beaker and mix with the stir bar. Use potassium hydroxide to adjust the pH to 5.8.Then dilute the solution with 500 milliliters of distilled water and transfer the diluted solution to a medium bottle. After this, autoclave the solution. Prepare the sterilization solution according the the text protocol.Then, working under aseptic conditions, place about 50 transgenic A.thaliana seeds carrying a fluorescent marker into a 1.5 milliliter tube. Next add one milliliter of 70%ethanol to the tube and mix by inverting the tube five times. After allowing the seeds to sink to the bottom of the tube, gently remove the ethanol with a micropipette.Add one milliliter of sterilization solution to the seeds and invert the tube five times to mix. Gently remove the sterilization solution from the seeds with a micropipette. Then add one milliliter of sterile water.Add 1.5 milliliters of the Murashige-Skoog solution to each well of a 24 well plate. Then add two sterilized seeds to each well. Using two layers of parafilm, tape the lid to the plate.Next, transfer the plate to a growth chamber to incubate for 14 days. First, transfer 30 microliters of Murashige-Skoog solution from the 24 well plate to the center of a glass slide. Using dissecting scissors, remove the cotyledon from a 14-day old seedling.Float the cotyledon with the observation side facing up on the drop of Murashige-Skoog solution. Prepare the cotyledon according to the methods previously described in the Journal of Visualized Experiments. Set the specimen on the stage of a confocal laser microscope.Finally, use bright field illumination to select clustered guard cells for observation. In this protocol, stomatal clustering was induced in A.thaliana seedlings. The clustered guard cells grown in the sucrose containing medium having larger chloroplasts than guard cells grown in sucrose-free conditions.Enlargement of the chloroplasts was confirmed with a chloroplast stroma marker and chlorophyll autofluorescents, suggesting that sucrose treatment resulted in starch grain accumulation in the chloroplasts. Additionally, confocal observation of GFP tub-6 revealed that the cortical microtubules were radially oriented in the sucrose-treated cells, just like in the sucrose-free guard cells. Actually we discovered this method by accident.After the finding, we examined the environmental response of clustered stomata. We believe our system could help to examine the significance of the stomata distribution.

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Spotting Cheetahs: Identifying Individuals by Their Footprints

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Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
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Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

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.

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.

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

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

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 ...