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.

Research

JoVE Journal

Biology

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