Name: a labor-saving and repeatable touch-force signaling mutant screen protocol for the study of thigmomorphogenesis of a model plant arabidopsis thaliana
Uploaded: 2019-08-06T13:00:00
Description: Watch this Scientific Journal Video about A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana at JoVE.

This study was supported by the following grants: 31370315, 31570187, 31870231 (National Science Foundation of China), 16100318, 661613, 16101114, 16103615, 16103817, AoE/M-403/16 (RGC of Hong Kong). Authors would like to thank Ju Feng Precision and Automation Technology Limited (Shenzhen, China) for their offering of several schematics shown in Figure 1.
Authors would also like to thank S. K. Cheung and W. C. Lee for their contribution to the development of the touch-force loading machine.

1. Seed preparation
NOTE: Arabidopsis seeds of both wild type (Col-0) as well as mkk1 and mkk2 loss-of-function mutants used were purchased from the Arabidopsis Biological Resource Center (ABRC, https://www.arabidopsis.org, Columbus, OH).
<ol>
	<li>Calculate how many plant individuals of each genotype will be used for a reliable statistical analysis. Prepare a sufficient number of seeds based on the germination rate of each line, usually 4-5 times more than what is...

<ol>
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H., Braam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+touch-and+darkness-regulated+Arabidopsis+genes:+a+focus+on+calmodulin-like+and+XTH+genes.">Genome-wide identification of touch-and darkness-regulated Arabidopsis genes: a focus on calmodulin-like and XTH genes.</a> New Phytologist. 165, 429-444 (2005).</li><li>Wang, K., 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=Quantitative+and+functional+posttranslational+modification+proteomics+reveals+that+TREPH1+plays+a+role+in+plant+touch-delayed+bolting.">Quantitative and functional posttranslational modification proteomics reveals that TREPH1 plays a role in plant touch-delayed bolting.</a> Proceedings of the National Academy of Sciences United States of America. 115, 10265-10274 (2018).</li><li>Hamilton, E. 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Thigmomorphogenesis is a complex plant growth response towards mechanical perturbations, which involves a network of cellular signaling and action of phytohormones. It is a consequence of adaptive evolution of plants to survive under the undesirable environmental conditions25,26. Mechanical touch, especially human finger touch and hand-held cotton swab touch, have been selected to study this morphological changes in previously thigmomorphogenetic studies<sup clas...

Plant thigmomorphogenesis is a term that was coined by Jaffe, MJ in 19731. It is a plant tropism but different from the well-known phototropism or gravitropism caused by stimuli of sunlight or gravity2,3. It describes phenotypic alterations associated with periodic mechanical stimulations, which have been frequently observed by botanists in earlier times4,5. Raindrops, wind, plant, animal and human touches, even animal bites, are all considered to be different types of mechano-stimuli that trigger the force signaling in plants4,5. Characteristics of plant thigmomorphogenesis include the delay of bolting, a shorter stem, smaller rosette/leaf size in herbaceous plants, and thicker stem in woody plants6,7,8. This is unlike the thigmonastic or thigmotropic response often found in the Mimosa plant or other mechano-sensitive vines, where these rapid touch responses are easier to be observed1,9,10. Thigmomorphogenesis, on the other hand, is relatively difficult to be observed because of its slow growth response. Thigmomorphogenesis is usually observed following weeks or even years of continuous force-loading stimulation. This unique nature of plant touch response makes it difficult to perform a forward genetic screen using human hand touch stimulation to isolate the touch-force signaling resistant mutants in a robust manner.
To elucidate the force signal transduction pathways and the molecular mechanisms underlying the thigmomorphogenesis6,11, molecular and cellular biological experiments have been performed in the past6,12,13,14. These studies have proposed that the plant force signal receptors mainly consist of mechanosensitive ion channels (MSC) and the tethered MSC complexes composed by multimeric complexes of membrane-spanning proteins11,14,15. The cytoplasmic Ca2+ transient spike generated within seconds of the initial touch. Wind-, rain-, or gravi-stimulation may interact with the downstream calcium sensors to transduce the force signals to nuclear events14,16,17,18. In addition to molecular and cellular studies, the forward genetic screen with manual finger touching of plants has found that phytohormones and the secondary metabolites are involved in the consequent touch-inducible (TCH) gene expression following the touch-force loading13,19. For examples, aos and opr320 mutants have been identified thus far from the genetic studies. However, the major problem associated with application of the forward genetics in the study of thigmomorphogenesis is still the intensive labor required for quantitating the level of touch response and touching a large population of genetically mutated individual plants. The time-consuming issue also persists in the hand touching-based mutant screen14,20. For an example, to complete one round of touch-force stimulation, a person needs to touch 30-60 times (one touch per second) on an individual plant. In order to have enough number of plants for statistical phenotype analysis, 20-50 individual plants of the same genotype are normally required for the touch-force loading process. This touch-force loading regime means that a person needs to repetitively perform 600-3,000 touches on one genotype of choice. This type of touch normally needs to be repeated 3 to 5 rounds a day, which equals roughly 1,800-15,000 finger or cotton swab touches per day per genotype of plants. A well-trained person is normally required to maintain the strength and force of multiple touches within a desirable range throughout many rounds of repetition in a day to avoid the large variation in force and strength. As it is well known that thigmomorphogenesis is a saturable and dose-dependent process6,21, touch force/strength becomes critical to a success in triggering touch response of a plant.
To remove the person-dependent touch-force loading and to maintain mechanical application within an acceptable error range14, we therefore designed an automatic touch-force loading machine to replace the hand-manipulated touches. The machine has 4 moving arms built, each of which is equipped with one human hair brush. This version is named Model K1 to specify its feature of human hair touch-force loading. If 4 genotypes are measured quantitatively for their thigmomorphogenesis or touch response under one machine, 40-48 individuals per genotype can be measured. Each round of touch repetition (less than 60 times of touch per plant) lasts less than 5 minutes using a moving speed adjustable robotic arm. Thus, plants on a Model K1 touch machine can be mechanically stimulated for multiple rounds a day either with a constant touch-force loading or different levels of strengths as initially programmed.
Arabidopsis thaliana, a model plant organism, was therefore chosen as the target plant species for testing the fully automatic hair touch-force loading machine application. Because there are several large seedbanks available for retrieving the various germplasms of mutants and the size of flowering, Arabidopsis fits well to the space available in the growth shelf mounted with the Model K1 touch machine.
The Model K1 automatic touch machine consists of three major components: (1) the H-shape metal rack composed by two belt-driven linear actuators, (2) robotic metal arms equipped with hair brushes, and (3) a controller. For a customized Model K1 touch machine, each X/Y axis module is composed of one belt-driven guide-rail, two slide blocks (red) and one 57 stepper motor (pre-installed and dismountable) (Figure 1A,B). The upper horizontal actuator allows the robotic metal arm to move left and right horizontally, the lower vertical belt-driven linear actuator allows the robotic metal arm to move up and down vertically (Figure 1B, Figure 2A). Four dismountable robotic arms were installed on the vertical actuator (Figure 1C, Figure 2B). Four human hair brushes were bound to four robotic arms, respectively (Figure 1C, Figure 2B). All mechanical parts to construct the Model K1 touch machine in bolded font below are marked in Figure 1C (also see the Table of Materials).

<table border="0" cellpadding="0" cellspacing="0" style="width:1141px;" width="1140">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">4 hair brushes</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">customized</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">4 robot arms with one holder</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">customized</td>
			<td>1000 mm length holder and 560 mm length robot arm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">57 stepper motor</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">57HS22-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">All purpose potting soil</td>
			<td style="width:393px;">Plantmate, Hong Kong</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Arabidopsis plant seeds</td>
			<td style="width:393px;">Arabidopsis Biological Resource Centers, Columbus, OH</td>
			<td style="width:149px;"></td>
			<td>For arabidopsis seed purchase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">BIO-MIX potting substratum</td>
			<td style="width:393px;">Jiffy Products International BV, the Netherlands</td>
			<td style="width:149px;">1000682050</td>
			<td>Two soils were mixed together to grow Arabidopsis. The ratio of All purpos potting soil and&#160; BIO-MIX is 1:2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">IL 1700 research radiometer</td>
			<td style="width:393px;">International Light, Newburyport, MA</td>
			<td style="width:149px;"></td>
			<td>The light intensity of both full-wavelength and photosynthetic active radiation can be measured.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">ImageJ</td>
			<td style="width:393px;">https://imagej.nih.gov/ij/download.html</td>
			<td style="width:149px;"></td>
			<td>Free downloaded software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Ju Feng Precision and Automation Technology Limited</td>
			<td style="width:393px;">Shenzhen, China</td>
			<td style="width:149px;"></td>
			<td>For belt-driven linear actuators and other mechanical modules purchase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Junction plate of the slide block</td>
			<td style="width:393px;"></td>
			<td style="width:149px;"></td>
			<td>To fix the Y guide-rail module or Y auxiliary girder onto backs of slide blocks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Junction plate of the X axis module</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">customized</td>
			<td>To connect the X guide-rail module and X auxiliary girder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Slide block</td>
			<td style="width:393px;"></td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">WDT4045 X axis guide-rail module</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">843 mm, customized</td>
			<td>Pre-installed with two slide blocks and one 57 stepper motor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">WDT4045 Y axis guide-rail module</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">1038 mm, customized</td>
			<td>Pre-installed with two slide blocks and one 57 stepper motor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">X axis auxiliary girder</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">843 mm, customized</td>
			<td>Pre-installed with two slide blocks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Y axis auxiliary girder</td>
			<td style="width:393px;"></td>
			<td style="width:149px;">1038 mm, customized</td>
			<td>Pre-installed with two slide blocks</td>
		</tr>
	</tbody>
</table>

a labor-saving and repeatable touch-force signaling mutant screen protocol for the study of thigmomorphogenesis of a model plant arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

The automatic hair touch-force loading machine 
For observation of morphological changes on plants, both the reproducible growth conditions and treatment methods are key to obtaining repeatable results. This high-throughput and automatic touch-force signaling mutant screening is achieved by the newly built hair touch-force loading machine, Model K1 (Figure 1, Figure 2). These hair brushes can touch a maximum of 4 trays of plants simultaneou...

Watch this Scientific Journal Video about A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana at JoVE.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

We build a automatic touch force machine to observe the plant signal morphogenesis. The robotic arms I installed with human hair brushes to apply gentle touch force on plants. In comparison with human finger touch, the automatic human hair touch machine can offer more labor saving, a uniform touch force loading.The time required for one round of force loading can be restricted within several minutes. As compared to a several hours of finger touch and a cotton swab touch, the robotic machine can largely improve the plant mercantile response research. By increasing the efficiency and population of treated plants, we anticipate that the automatic touch machine will be applied in a four signaling mutant screens and the mercantile response studies in both plants and animals.To begin, test the germination rate of each genotype after nurturing seeds on a 8%agar plate with MS medium for seven days. According to the germination rate, prepare seeds four to five times more than the desired number of plants to be produced. Immerse seeds in cold water in 1.5 milliliter plastic tubes and cover with aluminum foil to keep in the dark.Store at four degrees celsius for seed imbibition. Five to seven days after the imbibition, select the appropriate soil for plant growth. Avoid large clumps and use a fork to mix them homogeneously.Prepare 24 207 milliliter plastic cups with an upper rim diameter of 7.4 centimeters. Drill three round holes at the bottom of the cups for irrigation purposes. Fill these plastic cups with mixed soil.Let the soil pile up to around one centimeter higher than the cup rim and use a ruler to flatten the surface of the piled soil softly. Transfer 24 cups into a plastic tray. Set the light intensity of the growth chamber between 180 and 240 microeinsteins per second per square meter, which is higher than the photosynthetic active radiation for fast growth of the plants.Set the light condition to be 24 hours constant. Set the temperature of the growth chamber at a 23.5 plus or minus 1.5 degrees celsius and humidity between 35 and 45%Add 2.5 liters of water into each tray. Let the soil absorb the water from the holes located at the bottom of the cups and wait for the surface of the soil to drop to the cup rim level.Two hours later, sow three to four seeds into a single spot and four evenly distributed spots within a cup. Place a transparent plastic cover above each tray and let seeds germinate for a week. Then, remove the cover and allow seedlings to grow for another week.Irrigate plants with 1.5 liters of water every other day after the seeds germinate. Nine to 10 days after seed sowing, remove extra shoots by thinning. Use tiny scissors to cut the chutes and avoid pulling the entire plant out of the soil directly.Three to four plant individuals of similar size in each cup. To install touch hair brushes onto the robotic arms, first use glue to fix one layer of human hair evenly onto a 330 millimeter long steel ruler. The length of the hair is 126 millimeters.With two metal clamps, attach those steel rulers onto the robotic arms. In the control panel, set the height of the machine arms along the vertical dimension by pressing Jog F plus to raise and Jog R minus to lower the robotic arms and brushes. Keep the tip of hair brushes 5 centimeters lower than the cup rim.Press the Inc F plus or R minus to prerun the machine one to two cycles to make sure all plant individuals are being touched. Before the official experiment, use an electronic scale to measure the touch force. Adjust the height of the machine arms to maintain the touch force between one to two millinewtons.Next, set the starting position of machine arms along the horizontal dimension manually. Allow the hair brushes to hang at the edge of each tray. Press Jog F plus or Jog R minus to move the machine arm horizontally little by little to make sure that no plant is being touched before the touching experiment starts.Set the hair brush traveling distance in the horizontal dimension to 365 millimeters by pressing the travel button. Press Inc F plus or R minus to move the machine arms to obtain a full travel distance and ensure that all of the treated plants are being touched. Then, press the auto speed button to set the movement speed along the x axis of the machine arms at 5000 millimeters per minute.Press the minor cycle button to set the touch time at 20 trials equaling 40 touches per round. Set the repetition interval of the touch round at 480 minutes per day by pressing the major period button which allows hair brushes to touch plants for three rounds a day with an interval time of eight hours. The machine will start a new round of touch automatically when the countdown turns to zero.Set the major cycle at 12 trials to allow the machine to touch plants for 12 rounds within a period of four days. Press the start button to initiate the preset program. Adjust and calibrate the brushes and hair tips to the same height everyday during the entire touching period.To track the bolting day of each plant within a touching experiment, observe the first inflorescent stem of each plant everyday. Measure the length and record the duration of growth when it reaches one centimeter, meaning it has transitioned from the vegetative phase to the reproductive phase. The measure of rosette radius, take photos of the whole tray from the top.Use an image processing software to measure the distance from the rosette center to the tip of the longest leaf with the cup rim as a ruler. To measure rosette area, first, remove the inflorescence without affecting the rest of the rosette organs. Take photos from the top of each plant together with a scale ruler placed nearby.In the image processing software, measure the horizontal two dimensional surface area of the rosette leaves. In this experiment cotton swab manual touch treatment was compared with machine driven hair touch treatment on wild type control Columbia zero plants. Bolting delayed 1.7 days after continuous cotton swab touch treatment.Similarly for the automatic machine driven hair touch, delayed bolting was observed. The univariate Cox hazards analysis shows the bolting risk of plants in the touch group is 31%and 52%compared with plants in the control group, respectively. However, this bolting delay was not observed on tDNA insertional mutants, NKK one and NKK two.The univariate Cox proportional hazards model shows only wild type Columbia zero exhibited a significant difference between control and touch plants with an estimated HR of 41. For morphological changes, the wild type Columbia zero plant showed significantly smaller rosette radius and shorter leaf length after three days of constant and repetitive automatic machine driven hair touch. The projected rosette area was changed from 20 to 16 square centimeters after 13 days of touch.To make sure that the touch force loading will not lead to the wounding, appropriate touch force needs to be set carefully. A set of proper growth conditions are needed to produce a suitable Arabidopsis plant for touch response assay. For example, 24 hours of light intensity between 180 and 240 microeinstein per square meter per second is needed to observe the signal morphogenesis.To extend to the other forms of mechanical force loading, the machine can also be modified with water sprinkling nozzle or wind blowers to mimic rain or wind effects, respectively in the future.

a-labor-saving-repeatable-touch-force-signaling-mutant-screen

Research

JoVE Journal

Biology

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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 ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...