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

Summary

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.

Abstract

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’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.

Introduction

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 gravity^2,3. It describes phenotypic alterations associated with periodic mechanical stimulations, which have been frequently observed by botanists in earlier times^4,5. 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 plants^4,5. Characteristics of plant thigmomorphogenesis include the delay of bolting, a shorter stem, smaller rosette/leaf size in herbaceous plants, and thicker stem in woody plants^6,7,8. 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 observed^1,9,10. 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 thigmomorphogenesis^6,11, molecular and cellular biological experiments have been performed in the past^6,12,13,14. 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 proteins^11,14,15. 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 events^14,16,17,18. 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 loading^13,19. 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 screen^14,20. 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 process^6,21, 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).

Protocol

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).

1. 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 needed for an experiment. Ensure enough number of healthy and uniform-sized plants can be used for touch response assay. According to this protocol, 300-500 seeds per genotype are usually used to produce 80-90 plants of similar size.
2. Immerse seeds in cold water and store them in 4 °C (covered with aluminum foil to keep in dark) for seed imbibition. Sow the seeds 5-7 days after the imbibition.

2. Plant growth

1. Select the appropriate soil for plant growth (see the Table of Materials). Avoid large clumps and mix them homogenously.
2. Prepare 24 plastic cups: the holding capacity is 207 mL and the upper rim diameter is 7.4 cm. Drill three round holes at the bottom of a cup for irrigation purpose.
3. Fill these plastic cups with the mixed soil. Let the soil piling up to 1-2 cm higher than the cup rim and flatten the surface of piled soil softly.
4. Transfer 24 cups into a plastic tray (21 inches x 10.8 inches x 2.5 inches) and place the tray under constant light condition (see below).
5. Add 2.5 L of water into each tray two hours before seed sowing. Let the soil to absorb the water from holes located at the bottoms of cups and wait for the surface of the soil to drop to the cup rim level.
6. Sow 3-4 seeds into a single spot, and 4 evenly distributed spots within a cup.
7. 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.
8. Remove extra plants by thinning and keep 4 plant individuals of similar size in each cup 9-10 days after seed sowing.
9. Irrigate plants with 1.5 L of water every other day after the seeds germinate.

3. Growth condition

1. Set the temperature of the growth chamber at 23.5 ± 1.5 °C, and humidity between 35 and 45%.
2. Set the light intensity between 180 and 240 μE∙m^-2∙s^-1 (measured by IL 1700 research radiometer, International Light)¹⁴. The photosynthetic active radiation is from 90 to 120 μE∙m^-2∙s^-1.
3. Set the light condition to be 24 h constant.

4. The construction of touch-force loading machine

NOTE: This robotic hair touch-force loading machine (Model K1) is designed to serve purposes of both touch-force signaling mutant screening and plant thigmomorphogenesis generation (Figure 1, Figure 2).

1. Pre-installation modules (dismountable, Figure 1C)
   1. Install two slide blocks (I) and one 57 stepper motor (II) onto the X/Y axis guide-rail module (III/V).
   2. Install two slide blocks (I) onto the X/Y axis auxiliary girder (IV/VI).
2. Installation of other mechanical parts (Figure 1C)
   1. Fix the X axis guide-rail module (III) and X axis auxiliary girder (IV) together by assembling two junction plates (VII) at each end of the guide-rail.
   2. Fix the Y axis guide-rail module (V) onto the dorsal of two slide blocks (X axis) in a crossing position by assembling two junction plates (VIII) in between.
   3. Fix the Y axis auxiliary girder (VI) onto the dorsal of the other two slide blocks (X axis) in a crossing position by assembling two junction plates (VIII) in between.
   4. Assemble the holder of robot arms (IX) onto the front of two slide blocks (Y axis) in a crossing position with a junction plate (Figure 2A).
   5. Assemble 4 hair brushes (X) onto robot arms (IX) with clamps (Figure 2B).

5. Touch-Force loading machine setting

NOTE: All of controlling parameters to set the Model K1 touch machine in bolded font below are shown in the control panel (Figure 2F).

1. Install touch hair brushes onto the robotic arms. Use a 330 mm-long steel ruler as a holder to fix one layer of human hair (3,600-4,600 hairs/brush) evenly. The length of the hair is 126 mm (Figure 1C).
2. Fix those steel rulers onto the robotic arms with two metal clamps.
3. Set the height of machine arms along the vertical dimension (Y axis) first. Press Jog F+ to raise and Jog R- to lower the robotic arms and brushes. Let the tip of hair brushes 0.5 cm lower than the cup rim. Press the ZERO set. Pre-run the machine 1-2 cycles to make sure all plant individuals are being touched. Adjust and calibrate the brushes and hair tips to the same height every day during the entire touching period.
4. Use an electronic scale to measure the touch force (vertical loading) and maintain the touch force level at 1-2 mN¹⁴.
5. Set the starting position of machine arms along the horizontal dimension (X axis) manually. Allow the hair brushes to hang at the edge of each tray and make sure that no plant is being touched before the touching experiment starts. Press Jog F+/Jog R- to move the machine arm horizontally little by little to set the starting position.
6. Set the hair brush traveling distance in the horizontal dimension (X axis) to 365 mm by pressing the Travel button. Press Inc. F+/Inc. R- to move the machine arms to obtain a full travel distance and ensure that all of the treated plants are being touched during the entire touching experiment.
7. Set the movement speed along the X axis of the machine arms at 5,000 mm/min by pressing the Auto Speed button. Keep the same movement speed during the entire touching experiment.
8. Set the touch time at 20 trials by pressing the Minor Cycle button. Keep the same number of touches per round during the entire touching experiment.
   NOTE: One Minor Cycle equals two Travel distances, which means machine arms will move from the starting position to the end position and then back to the starting position. One minor cycle generates two touches. Hair brushes touch plants 40 times within 20 trials (2 touches x 20 trials = 40 touches). The 40-touch is defined to be one round of touch-force loading.
9. Set the repetition interval of the touch-round at 480 min per day by pressing the Major Period button. Keep the same frequency of touch rounds during an entire touching experiment.
   NOTE: This allows hair brushes to touch plants for 3 rounds a day, and the interval time between each round is 480 min (8 h). The displayed blue number stands for the interval time of each touch round. The machine will start a new round of touch automatically when the countdown below (red number) turns to 0000.
10. Set the Major Cycle at 12 trials, which means that the machine will touch plants for 12 rounds within a period of 4 days automatically. This setting of 12 trials is used to avoid human error in skipping a day of touching.
11. Press the start button to initiate the pre-set program. The Model K1 touch machine will automatically perform the touch force loading according to settings.

6. Physiological data collection and analysis

1. Days to Bolting: Record the bolting day of each plant individually within a touching experiment. Bolting is a symbol that a plant changes its growth stage from the vegetative phase to the reproductive phase. In Arabidopsis, the bolting day is defined as the number of days used by a plant to have its first inflorescence stem reach 1 cm in length.
   NOTE: Under the growth condition described above, the bolting of wild type plants normally initiates from 19 to 23 days after seed sowing and ends at 28-32 days.
2. Rosette Radius: Measure the distance from the rosette center to the tip of the longest leaf.
   1. Take photos of the whole tray from the top. Take photos of the control group and the touch-treated group separately.
   2. Download the appropriate software. Use the free downloaded software ImageJ (https://imagej.nih.gov/ij/download.html) for example.
   3. Open a photo file, use the zoom function to zoom the photo into an appropriate size.
   4. Choose the Straight tool to draw a straight line between the rosette center and tip of a longest leaf to measure the rosette radius.
   5. Select one plant and press the left button to draw a straight line from the rosette center to the longest leaf tip.
   6. Choose the Analyze-Measure function or press Ctrl + M to analyze the line distance.
   7. Select one cup and repeat the previous two steps to analyze the diameter of each plastic cup at the same time. Use these data to perform the calculation to eliminate the bias resulting from the photo-taking.
      NOTE: The equation is:
      R_a/D_a = R_m/D_m
      (R_a, the actual Rosette Radius of a plant; D_a, the actual diameter of plastic cup; R_m, the measured Rosette Radius of the same plant determined by a software; D_m, the measured diameter of the plastic cup which is used for growing the same plant)
3. Rosette Area: Measure the horizontal 2-dimensional surface area of rosette leaves.
   1. Remove the inflorescence without affecting the rest of rosette organs.
   2. Take photos from the top of each plant together with a scale ruler placed nearby.
   3. Use one free plugin of ImageJ, Rosette Tracker and follow the protocol published previously²².

Representative Results

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 simultaneously. There were 24 cups placed in a tray, and 12 cups of plants in a group used as both control and the treated plants (Figure 2C,D). In each cup, four plants were grown and a total of 48 or less plant individuals were touched by the same hair brush, which guarantees enough plants for later statistical analysis. A maximum of 4 genotypes of plants can be touch-treated simultaneously on one Model K1 touch machine. One of the key points is the setting of the touch machine arm/hair height because thigmomorphogenesis is dose-dependent^6,21. Different hair positions with respect to the plant rosette leaf position generate different touch forces, which may generate totally different thigmomorphogenesis results. In our experiments, the plant-contacting tip of hairs should be placed 0.5 cm lower than the cup rim (Figure 2E), which generates forces that are similar to the previously published touch force¹⁴. A programmable controller installed into a touch panel is used to control the whole touch-force loading machine (Figure 2F, see Table of Materials).

The comparison of two different touch methods
To compare this automatic machine-driven hair method with the conventional manually cotton swab touching method, two independent experiments were performed on Col-0 (Figure 3). In the cotton swab touch group, touching started from 12-day-old plants. Each round had 40 touches (1 touch/s). In total, 3 rounds were performed each day (Figure 3A). It showed 1.7 days of delay in bolting after a continuous cotton swab touch treatment (22.1 ± 0.2 days vs. 23.8 ± 0.2 days). Similarly, for the automatic machine-driven hair touch, the touch-force loading initiated from 14-day-old plants and 40 times of touch (within 3 min) were applied for a round. In total, 3 rounds of touches were performed a day with exactly 8 hours of interval (Figure 3B). Delayed bolting was observed for Col-0 plants. The average bolting time was 23.0 ± 0.3 days, while the bolting time of the Model K1 touch machine-treated plants was 24.7 ± 0.2 days. The differences between control and touch-treated plants were consequently analyzed with the univariate Cox proportional-hazards model. It offered the estimated Hazard Ratio (HR) of 0.31 (cotton swab touch) and 0.52 (machine-driven hair touch), respectively (Figure 3C), which means that the bolting risk/probability of plants in the touched group is 31% and 52% compared with plants in the control group, respectively. This indicates that the possibility of bolting of the touched wild type plants is around half as compared to that of the untouched control plants regardless of whether it is manual touch with a cotton swab or the automated hair touch.

The prospective results on different touch mutants
Recent preliminary data suggested that MKK1 and MKK2 might play an important role in the touch response of Arabidopsis¹⁴. We selected these two mutants and conducted touch experiments on these putative touch response mutants using the automatic hair touch-force loading machine (Figure 4, Table 1). The wild type control plants showed 1.8 days of bolting delay (24.1 ± 0.3 days vs. 25.9 ± 0.2 days, Figure 4A) just as the previous report¹⁴ while this bolting delay was not observed on T-DNA insertional mutants, mkk1 (24.6 ± 0.2 days vs. 24.4 ± 0.3 days, Figure 4B and Table 1) and mkk2 (23.9 ± 0.1 days vs. 24.2 ± 0.2 days, Figure 4C and Table 1). By analyzing these data with the univariate Cox proportional-hazards model, only wild type Col-0 exhibited a significant difference between control and touched plants with an estimated HR of 0.41 (Figure 4D). These touch-force loading experiments conducted by the automatic hair touch-force loading machine demonstrated that mkk1 and mkk2 mutants are touch response mutants.

The measurement of other morphological indexes
Morphological changes associated with thigmomorphogenesis are not limited to the delaying of bolting. Both shorter stem and smaller rosette leaf size are also the components of thigmomorphogenesis^6,7,9,14. Hence, we reported here two additional types of measurements on morphological indexes of touch response, rosette radius/leaf length and rosette (projected) area (Figure 5). Similar to the previously observed phenotype change, the wild type Col-0 plant showed significantly smaller rosette radius and shorter leaf length after 3 days of constant and repetitive automatic machine-driven hair touch (1.77 ± 0.05 cm vs. 1.50 ± 0.04 cm, Figure 5A). The projected rosette area was changed from 20.32 ± 0.53 cm² to 16.19 ± 0.48 cm² after 13 days of touch (Figure 5B). Both mkk1 and mkk2 had the similar reduced rosette radius and area. Taken together, these data demonstrated that MKK1 and MKK2 proteins are important for the bolting delay of Arabidopsis and not required in shaping the rosette size and rosette area.

Statistical Analysis
As to the box and whisker plots shown in Figure 2 and Figure 3 and the column charts shown in Figure 5, the statistical significance was analyzed by two-tailed student’s t-test, with significance represented by *** and n.s. at p < 0.001 and p > 0.05, respectively. For the Kaplan-Meier plots shown in Figure 2 and Figure 3, a univariate Cox hazard analysis was used to analyze the effect of touch treatment on the bolting event^23,24. The Hazard Ratio (HR), 95% Confidence Interval (95% CI) and p value are offered in the tables below. For instance, HR = 0.5 means that on a specific day, the bolting risk/probability of plants in the touched group was 0.5 or 50% compared with those plants in the control group.

Figure 1. The construction and parameters of the automatic hair touch-force loading machine. (A) Default schematics of the linear actuator. The upper left panel is the lateral view and the lower left panel is the dorsal view. The total lengths of the X axis module and Y axis module are 843 mm and 1,038 mm, respectively. Each default X/Y module is composed of one guide-rail, one slide block and one 57 stepper motor (pre-installed and dismountable). For a customized Model K1 touch machine, each X/Y module is composed of two slide blocks (red). The junction plate of the X module is enlarged from 56 mm to 100 mm to offer better connection and support. The upper right panel is the cross-section of the guide-rail and the lower right panel is the 57 stepper motor. (B) Schematics of the constructed double X axis and double Y axis linear actuators. This is the major part of the touch-force loading machine. The lower left panel is the dorsal view of constructed linear actuators. The upper left panel is the lateral view of the X axis module (843 mm). The middle panel is the lateral view of the Y axis module (1,038 mm). The upper right panel is the dorsal view of 4 slide blocks on the Y module and Y auxiliary girder. The lower right panel is the dorsal view of the junction plate on the X module. (C) The flowchart of machine part assembly. Different parts are marked and named in the figure. Detailed assembling processes were described in the protocol. The unit shown is this figure is mm. Please click here to view a larger version of this figure.

Figure 2. The overall design of the automatic hair touch-force loading machine. (A) The finished Model K1 touch machine. The photo was taken from the front side. The upper linear actuator controls the robot arm moving horizontally and the lower linear actuator controls the robotic arm moving vertically. (B) The lateral view showing dismountable robotic arms. Hair brushes were clamped onto the robotic arms. (C and D) Photos showing how human hairs brushes touch the plants, which were taken from the front side and the lateral side, respectively. (E) The lateral view showing how to set the height of the hair brush against the cup rim. Both the machine arms and hair brushes are visible. (F) The operation interface of the Model K1 touch machine. A programmable controller (AFPX-C30T) linked to a touch panel (MT6070i) is used to control the whole machine. Detailed settings and operating procedures are described in the protocol. Please click here to view a larger version of this figure.

Figure 3. Comparing the effects of two touch methods on thigmomorphogenesis. (A-B) The comparison of manual cotton swab touch (A) and human hair touch driven by the automatic touch-force loading machine (B), respectively. Box and whisker plots are shown in the left panel, which show the comparison of average bolting day between the control group and the touched group. Means ± SE are shown. Statistical analysis was performed by a Student’s t-test. Significance at p < 0.001 is shown as ***. Kaplan-Meier plots are shown in the middle, which are the percentage of bolting plants over the growth time (days after sowing). The right panel shows representative individuals of untouched control and touched plants that show the difference in bolting time and inflorescence stem height. (C) The summarized table: the numerical numbers in control and touched columns are the plant number used for statistical analysis. The Hazard Ratio (HR), 95% Confidence Interval (95% CI) and p value under the section of univariate Cox hazard analysis are offered. The bolting risk and probability of plants in the touched group were 31% and 52% as compared to the untouched group, respectively. The univariate Cox hazard analysis was estimated by SPSS. Please click here to view a larger version of this figure.

Figure 4. The thigmomorphogenesis of mkk1 and mkk2 mutants as well as the wild type plant (Col-0) induced by the automatic hair touch. (A-C) The prospective touch response of Col-0 (A), mkk1 (B), and mkk2 (C) generated through repetition of human hair touches driven by the automatic touch-force loading machine. Box and whisker plots are shown in the left panel, which are the comparison of average bolting day between the control group and the touched group. Means ± SE are shown. Statistical analysis was performed by a Student’s t-test. The *** and n.s. represent p < 0.001 and p > 0.05, respectively. Kaplan-Meier plots are shown in the middle, which are the percentage of bolting plants over the growth period (days after sowing). The right panel shows representative individuals of the untouched control and the touched plants that show the bolting difference. Data of mkk1 (B) and mkk2 (C) were compiled from two and three biological replicates, respectively. Detailed plant numbers used in each replicate were shown in Table 1. (D) The summarized table: the numbers under control and touch columns were the plant number used/analyzed in these two groups, respectively. The HR, 95% CI and p value under the section of univariate Cox hazard analysis were offered. The bolting risk/probability of wild type plants in the touched group is 41% as compared with the control group. The univariate Cox hazard analysis was estimated by SPSS. Please click here to view a larger version of this figure.

Figure 5. The rosette radius and area measurement for defining thigmomorphogenesis. (A-B) The rosette radius and the rosette area of the wild type were measured at day 17 and day 27 after seed sowing, respectively. Bars shown in the upper left panel are the comparisons of either rosette radius or rosette area between the control group and the touched group, respectively. Means ± SE are shown. Statistical analysis was performed by Student’s t-test; ***p < 0.001. Photos shown in the upper right panel are representative individual plants. The summarized tables below show the plant number analyzed in the control group and the touched group. Both the rosette radius (cm) at day 17 and rosette area (cm²) at day 27 are also shown. Please click here to view a larger version of this figure.

Table 1. The bolting data of different biological replicates. The summarized table contains two biological replicates of mkk1 and three biological replicates of mkk2.

Discussion

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 conditions^25,26. Mechanical touch, especially human finger touch and hand-held cotton swab touch, have been selected to study this morphological changes in previously thigmomorphogenetic studies^14,20. This simplified version of touch-force loading to trigger plant touch response is easier to control and apply. In addition, this type of touch-force loading method can in some way mimic the wind- and rain drop-stimulated force signals produced in the natural environment¹⁹. The touch force is able to trigger calcium spikes, induce protein phosphorylation¹⁴ and the downstream gene expression mediating touch response¹⁹. Similarly, human hair brushes mounted on automated moving arms can also generate the plant touch response by mimicking the human hand-manipulated touches. To diversify the types of force application, water sprinkling nozzles and/or wind blowers can also be installed onto the robotic arms of the machine and used for a physiological experiment (Figure 2). The unique feature makes the automatic mechanical-force loading machine more versatile in the morphogenetic and physiological studies. The biggest advantage of this automatic mechanical-force loading machine is probably its labor-free, repeatable and time-saving feature, which makes it possible to perform a specific mutant phenotype selection from a large number of mutagenized individuals. In contrast to hours of human hand-manipulated touches, the Model K1 touch machine can touch various mutants simultaneously and complete a round of touch within 3 to 5 min. The time frame for a round of touch largely depends on the program setting at the beginning of treatment. If each individual plant would be touched 40 times in a round, the Model K1 machine would only need 9-15 min to finish three rounds of touch treatment within a day. The interval time between each round of touches can be precisely controlled; it is less likely for human beings to achieve such a precision.

Another important issue regarding the touch treatment is which stage of plant growth the touch force needs to be applied upon. In our practice, touching started 14 days after seed sowing for both the wild type and two mutants as the growth rates of these three genotypes are similar. For those mutants that have a significant difference in development time from the wild type, one may choose a different initial day to start the touching. Performing the one-way ANOVA test on the bolting data of both wild type plant and mutants for multiple comparisons can help¹⁴. This statistical analysis can offer the proper conclusion about the differences of bolting time generated by genotypes. In this case, a multivariate Cox proportional hazard analysis should be used to consider two variable parameters.

To set the touch-force level of the human hairs mounted on the Model K1 touch machine, we adjusted both the height (vertical force) and the speed (horizontal force) of the hair brushes (Figure 2E). The right settings were determined based on the preliminary data collected from many rounds of force level tests on an Arabidopsis plant placed on an electronic scale. As we have found, keeping both the hair height and the speed unchanged throughout the entire touch response experiment will produce a similar and constant thigmomorphogenetic phenotype among replicates for an Arabidopsis line. Too heavy a touch-force may kill the young seedlings as the fast moving hair brushes may lead to wounding on the surface of a leaf. In contrast, too light a touch-force may not be enough to trigger the delay of bolting within 2 weeks of repetition of touching. In our previous experiment, we have determined the appropriate touch-force loading to be 1-2 mN per touch^14,19. The hair length of 0.5 cm lower than the cup rim is used to generate a similar vertical touch force on Model K1 machine-based hair touch with a gentle horizontal moving speed 5000 mm/min (Figure 2E). This fixed setting of Model K1 machine reduces the variation of force strength resulted from the human error.

Overall, the hair touches performed by the automatic touch-force loading machine provide only an average touch-force loading on plants. The precise touch-force applied, especially the horizontal force loaded, is difficult to calculate either for a single hair or a group of hairs on a brush. In addition, the variance of plant shape and stem height can interfere with the application of horizontal force. Measuring this kind of physical strength or stress needs a more precise pressure sensor linked to a hair or a group of hairs. It is believed that more precise pressure sensor and mathematical modelling will be applied to improve the automatic touch-force loading machine in the future. The growth conditions, such as light intensity, moisture of soil and temperature of the greenhouse as well as nutrients supplying, all play a crucial role in the touch response phenotype development. Any stress conditions, such as drought, weak light condition with less than 90 μE∙m^-2∙s^-1, and a higher or a lower temperature that may affect the normal growth of Arabidopsis will interfere with the measurement of touch response of both wild type and mutants.

In short, this automatic touch-force loading machine can offer more labor-saving and uniformed average touch-force loading than human finger touch and cotton swab touch. It is expected that the Model K1 touch machine will be applied in various high-throughput touch-force signaling mutant screening and touch response analysis among agricultural crops or probably animal models with some modifications of the touch-force loading machine.

Materials

Name	Company	Catalog Number	Comments
4 hair brushes		customized
4 robot arms with one holder		customized	1000 mm length holder and 560 mm length robot arm
57 stepper motor		57HS22-A
All purpose potting soil	Plantmate, Hong Kong
Arabidopsis plant seeds	Arabidopsis Biological Resource Centers, Columbus, OH		For arabidopsis seed purchase
BIO-MIX potting substratum	Jiffy Products International BV, the Netherlands	1000682050	Two soils were mixed together to grow Arabidopsis. The ratio of All purpos potting soil and  BIO-MIX is 1:2
IL 1700 research radiometer	International Light, Newburyport, MA		The light intensity of both full-wavelength and photosynthetic active radiation can be measured.
ImageJ	https://imagej.nih.gov/ij/download.html		Free downloaded software
Ju Feng Precision and Automation Technology Limited	Shenzhen, China		For belt-driven linear actuators and other mechanical modules purchase
Junction plate of the slide block			To fix the Y guide-rail module or Y auxiliary girder onto backs of slide blocks
Junction plate of the X axis module		customized	To connect the X guide-rail module and X auxiliary girder
Slide block
WDT4045 X axis guide-rail module		843 mm, customized	Pre-installed with two slide blocks and one 57 stepper motor
WDT4045 Y axis guide-rail module		1038 mm, customized	Pre-installed with two slide blocks and one 57 stepper motor
X axis auxiliary girder		843 mm, customized	Pre-installed with two slide blocks
Y axis auxiliary girder		1038 mm, customized	Pre-installed with two slide blocks