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 ...

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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)

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 ...

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 ...