A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant–Environment Interactions

In this protocol, we referred to 4 L pots loaded on 20 cm x 20 cm scales, with each pot containing one plant. The same protocol is easily scalable and can be used with much bigger pots (up to 25 L loaded on 40 cm x 40 cm scales, with only a linear adaptation to the protocol measures) and several plants per pot. Thus the protocol can be easily adapted for plants of many types and sizes. Please refer to Figure 1 and Figure 2 for the system components.

1. Prepare the pots for the experiment

1. Insert the soil filter. Spread the nylon mesh (net) on top of the whole pot and place the net-holder on top of the net. With a hand, slowly push the net holder half way down the inside of the whole pot. Make sure that the net remains uniformly spread as it is pushed down between the two pots.
2. Insert the fiberglass stick (pole) between the two pots and push it all the way down to the bottom of the whole pot, making sure that it is on the outer side of the net as well and does not push the net.
3. Before pushing the net holder all the way down, push the net down by hand from inside the pot and adjust it so that it is spread uniformly and tightly over the bottom of the pot once the net holder has been fully inserted (Figure 2CI).
4. Slide the gasket ring from the bottom of the pot set-up described above, a third of the way up the side of the pot. Make sure that the slits of the ring open toward the bottom of the pot (Figure 2CII).
5. Repeat steps 1.1-1.4 for all of the experimental pots before continuing on to the next step. Randomize the location of the plants (Figure 2D; in either a randomized block design or a fully random design) using the Array Randomizer application.
   NOTE: To download the free program and for more information, please see the link: https://drive.google.com/open?id=1y4QbTpxRK5Lx430xzu1RFdrlcL8pz_1q).
6. Label the pots according to their locations in the array inside the greenhouse. For example, the label “B10D” corresponds to a pot located on Table B in Column 10 and Row D. Prepare three additional pots for each table for soil-water-content measurements (please see Section 7.1).

2. Grow the plants

1. Choose the growing (potting) medium that best suits the experiment. Choosing the right medium for the experiment is crucial and the correct choice depends on several factors (see Discussion). For first-time users, we strongly recommend using a porous, ceramic, small-sized medium. Please refer to Table 1 and Table 2 for more information to help choose the right medium for the experiment.
2. Germinate the seeds in cavity trays with the desired potting medium. If possible, do this inside the same greenhouse to be used for the main part of the experiment, in order to acclimate the plants to the environmental conditions inside that greenhouse.
3. If the seedlings were not germinated in trays, transplant them into cavity trays containing the potting medium. Plant one seedling in each cavity and let it grow until its roots are dense enough to take the shape of the cavity (root-soil plug).
4. Leave 5–7 cavities without seedlings for soil weight measurements (only potting medium; Figure 3). For more information, please see Section 5.9.

3. Improve the signal-to-noise level

NOTE: The following steps improve the quality of the measurements and reduce the noise levels.

1. Calibrate the lysimeter.
   1. Use a spirit level to check that all of the lysimeters are level and then start the weight calibration process. Use two standard weights (1–10 kg). Perform the calibration while the green container, including all plugs, is on the load cell.
   2. Put the first (lighter) calibration weight on each load cell.
   3. In the operating software, go to the Calibration tab and choose the weight for the first point. Then, select the load-cell position where the weight was placed and click Get point1 (Supplementary Figure 1A). This step can be applied on several load-cells simultaneously.
   4. Repeat for the second weight and click Get point2.
   5. Click Apply calibration.
2. Ensuring a sufficient quantity of plants with an appropriate size for the experiment
   NOTE: The smaller the plant, the weaker its signal will be (e.g., weight of water transpired in a day versus the pot weight). The following steps will help to improve the signal-to-noise ratio.
   1. Start the experiment when the plant transpires approximately 10% of the maximum pot water capacity.
      NOTE: For example, if working with a sandy medium that contains approx. 1 L of water at pot capacity (see Table 2), start the experiment when the plants transpire approx. 100 mL per day. If working with a peat-based medium that holds about 2 L of water at pot capacity (see Table 2), start the measurements when the plants transpire about 200 mL per day.
   2. Estimate the initial plant daily transpiration before loading it onto the system by measuring (manually) morning vs. evening weight differences in a few seedlings.
   3. When working with small plants, put several plants in each pot (e.g., six Arabidopsis plants in one 3.9 L pot²³, to reach the recommended minimal level of transpiration)*.

4. Setting up the experiment

NOTE: The process of setting up the experiment is designed to take into account the weight of all the parts of the system, namely, the weight of the potting medium (including the soil-water weight at pot capacity) and the initial weight of the seedlings. Follow the steps below:

1. If possible, work with similar static components that have similar weights. Static weight components include pot sets, soil probes and other plastic parts.
2. To start a new experiment, open the operating software. Open the Experiments tab in the menu on the left side of the screen. Click on Create New or duplicate the experiment properties from a previous experiment by right-clicking on the desired experiment and choosing Duplicate. Rename the experiment (Supplementary Figure 1B).
3. Make sure that no unit is being used in a different experiment currently running in the system. Check that all of the plants in the Plants table match the experimental design. If not, change the table according to the design (please see Sections 5.18, 6 and Supplementary Figure 1C).
4. Start the experiment by clicking on the experiment name and then clicking Start.
5. Take manual measurements of the pre-prepared empty pots (double pot, net, stick and black gasket ring). If using parts that are similar to one another, the average weight of 10 of them will be sufficient.
6. Mix the potting medium thoroughly with some water, for at least 1 h, so that it breaks down into homogeneous particles and is saturated, to ensure uniformity and homogeneity. For first-time users, we strongly recommend using a porous, ceramic, small-sized medium (see Table 1 and Table 2). As a second option, use coarse sand.
   1. Use a mechanical mixer (e.g., a concrete mixer).
   2. When a highly homogenous medium (i.e., industrial sand) is being used, skip step 4.6.1.
7. Fill all of the pots uniformly for the experiment with the appropriate potting medium (e.g., sand, soil or peat).
8. Insert a cast of a cavity mold (Figure 3B) that is similar in shape and size to the root-soil plug of the seedlings (from cavity tray) into the middle of the potting medium. Push it in completely. Tap the bottom of the pot against the floor a few times to make sure that the potting medium is well distributed in the pot. Repeat for all pots.
9. Water the pots well and rinse off the outside of the pots. Allow the pots to drain for 30 min before continuing on to the next step. Make sure that the pots drain freely. If the potting medium drains too slowly (e.g., dense peat), premix it with an airy substrate (e.g., perlite; please see also Table 1 and Table 2) to ensure faster drainage.
10. After the drainage has completely stopped, place all of the filled pots on the center of the lysimeter array (in the green containers that are already there) according to the experimental design (Figure 2A).
11. Check that the green containers are properly fitted into the load cell cover and not touching one another.
12. In the operating software, open the Experiment tab and select the Measure Components tab. Click on Measure object. Name the measurement “1^st measurement” (Supplementary Figure 1D).
13. Place the irrigation drippers, probes and pot covers on each pot. Make sure that the lines for the multi-outlet drippers and the probe cables are supported by their respective stands (attached to the units for each lysimeter scale; Figure 1E) before placing them in the pots. Make sure that all of the drippers, probes and covers are securely positioned.
14. Wait up to 3 min for a new measurement to be taken (data are collected automatically every 3 min) and then open the Experiment tab. Select the Experiment tab and click the Experiment. Meta-tag this measurement to the “1^st measurement” taken and name it “Static components” (Supplementary Figure 1E). Meta-tags are used when wanting to record a weight value that is determined by subtracting one measured value from another.
15. After making any necessary adjustments to the system, wait for a new data point to be recorded (every 3 min) before taking the next measurement.
16. Check the Static Components column to confirm that the values recorded in the Plants table do not include outliers. If any of the weights recorded are too low or too high, check for any interference with the load cell (e.g., make sure that nothing is touching it) and then take a new measurement (after the system was still for 3 min).
17. Click the Plants tab. Export the Plants table as a spreadsheet, add the average pot weight (from Step 4.5) to the measurement of the static components – “Tare weight”. Save and upload the file (import tab).
18. Make sure that all of the drippers are securely inserted into the potting medium and to the pipe coming from the controller. Back in the operating software, in the Experiment tab, select Treatments Scenarios. Click Create New to make a new “Plan”.
19. In the plan, choose the first step (create a new step if needed) and open it. Choose “Test” for Treatment and “Never” for Termination. In the step option, choose any treatment that is listed in the Irrigation Treatments tab above Experiments (Supplementary Figure 1F; please see also Step 4.21). Press the Apply tab.
20. Extract the Plants table as a spreadsheet, add “Plan” to the Treatment column and add “1” to the Step column. Save and upload the file.
21. Under the Irrigation Treatments tab, choose the “Test” treatment and set it to an irrigation time of 4–5 min [with the exact amount of time depending on the volumetric water content (VWC) of soil used] to enable drainage. Set the time 2 min ahead and go to the pots in the greenhouse. Other treatments can also be created. (See the detailed explanation in Step 7.4.)
22. Check visually that all of the drippers are working and that water is dripping out of the perforated drain plug of the green container.
23. In the experiment, change the irrigation treatment on Plan “X”, Step 1 (please see step 4.19-4.20) to the desired irrigation treatment. Make sure that each night irrigation (with fertigation; see Table 3 for the fertigation components used) is divided into several short pulses (events) with substantial pauses between them (at least three events every night), to ensure that the soil reaches its field capacity before dawn.
24. Let the irrigation program run for 1 or 2 days to let the soil reach its field capacity and continue on to the next phase.

5. Starting the experiment

NOTE: The data collected at this stage will be used as reference values for the rest of the experiment. Therefore, it is important to follow the next steps carefully.

1. Repeat Steps 4.18 through 4.20. Alternatively, start the process in the early morning, not long after the latest irrigation step.
2. Check visually that all of the pots are irrigated and that excess irrigation liquid is dripping out of the perforated drain plug of the green bath.
3. Remove the green, unperforated plug (from the lowest orifice) of the green container and let the water drain out completely. Then, put the plug back in its place (Figure 1D). If working on “drainage 0” (i.e., with the bottom hole open/the hollow drainage plug connected on the lowest hole), skip this step.
4. In the operating software, open the tab for the experiment and go to Measure Components. Click Measure Object and name the measurement as “Cast-pre”. Gently remove all of the casts from the pots and then wait 3 min for a new measurement to be recorded (Supplementary Figure 1D).
5. Click Measure Object, name it “Cast-post” and meta-tag the measurement to “Cast-pre”. The option will automatically calculate the difference between the two measured values and give the cast weight to verify the weight sensitivity.
6. Check the weight values in the Plants table. The difference between the “Cast-post” measurements should be no more than 20 or 30 g.
7. To measure the weight of the wet soil, in the operating software, go to the Measure Components tab in the experiment and select the Measure Soil Wet Weight option. Take the measurement by clicking OK when asked. Check the Soil Wet Weight measurements in the Plants table of yotheur experiment. The weight will appear in the “Soil Wet Weight” column (Supplementary Figure 1D,G).
8. If some of the measurements seem to fluctuate inappropriately, please do the following:
   1. Confirm that each pot is positioned correctly and is not touching any neighboring pot(s).
   2. Disconnect the first controller on the table from the electricity (the rest of the controllers are serially connected to one another and thus will shut down as well) for 2 min and then reconnect it.
9. Measure manually the average weight of a few (5–10) cavities with potting medium (from Step 2.3) without seedlings (soil plug). [Make sure that the soil plugs are well irrigated (i.e., to field capacity after drainage) before manual measurements.] In the Measure Components tab, press Set Seedling Bulk-Soil Weight and fill in the average weight (Supplementary Figure 1D).
10. Click Measure Plant Initial Weight. This first measurement is a reference point before planting (Supplementary Figure 1D).
11. Make sure that the seedlings in the cavity trays are well irrigated (i.e., to field capacity after drainage). Gently pull the seedlings with their root-soil plug from the cavities, making sure not to injure them, and place them carefully into the cavities made by casts in the pots, according to the experiment design. It is preferable to transfer the plants at dawn or dusk, in order to minimize the stress to the plants (i.e., to minimize wilting).
12. Wait for 3 min. Click Measure Plant Initial Weight again. This second measurement is the plant initial weight. Meta-tag the measurement to the first one (the reference point). The software will calculate the difference between the two measurements and subtract the Seedling Bulk-Soil Weight. The result is the plant initial weight.
13. Check the measured values in the Plants table of the experiment to make sure that they fall within a reasonable and logical range (Supplementary Figure 1C).
14. Saturate the soil by repeating Steps 4.18 through 4.20.
15. Make sure that all of the pots are draining properly. If not, repeat the saturation process. Wait 30 min for the drainage to cease. (See also Table 1 regarding the correct choice of potting medium.)
16. Under the Measure Components tab, click Measure Reserved Water Weight (Supplementary Figure 1D).
17. Extract the Plants table as a spreadsheet, subtract the measured Plant Initial Weight and Seedling Bulk-Soil Weight from the reserved water weight measurement (the “Reserve Water Inventory” column). Upload the file (Supplementary Figure 1C).
18. Confirm that the time period during which daily transpiration will be recorded is appropriate for the goals of the experiment. Fill in the values in the experiment general tab as appropriate for the project (Supplementary Figure 1H).
    1. Fill in zero hour: The time at which the software will check whether it needs to move to the next step in the treatment scenario.
    2. Fill in daily transpiration values: Daily transpiration is calculated as the difference between two weight windows during the day, for all days. The daily transpiration start time is the time at which the software will begin to measure the average weight.
19. Monitor the plants for 1–2 days before starting a new experiment (duplicate and rename the experiment).

6. Change the Plants Table

1. Extract the Plants table as a spreadsheet and change the table according to needs. Do not change the Plant IDs, Names or Positions. Save and upload the file.
2. Labeling (grouping) columns: To present or analyze (please see Step 8) grouped plants based on common labels (e.g., treatment, line), add a new column and label starting with # (e.g., #Treatment). In this column, make a notation for each plant (e.g., for “#Treatment” label, mark the plants as drought, control, etc.; Supplementary Figure 2).
   NOTE: The protocol presented above is the most advanced and comprehensive protocol for this system. However, first-time users may want to start with the simplified protocol (see Supplementary MS). The simplified protocol yields information about fewer traits and may lead to higher noise levels. But, at the same time, it provides a way to more easily become acquainted and familiar with the most important experimental procedures, hardware and software.

7. Run the experiment

1. Calculate the soil gravimetrical water content/soil water content (SWC value).
   NOTE: Gravimetric soil water content is different from volumetric soil water content (VWC).
   1. The SWC value is the ratio between the dry weight of the soil and the wet weight of the soil. To calculate SWC, use the three extra soil-filled pots (Step 1.3) without plants that were previously prepared and placed on a side table inside the greenhouse for a few days and irrigated regularly. Weigh the wet soil in an aluminum tray in the early morning, as soon as possible after the last irrigation event.
   2. Dry the aluminum tray with the soil in an oven (at 105 °C) for 4–5 days. Verify that the soil is completely dry by taking two consecutive weight measurements at least 60 min apart. If the weights are identical, the soil is indeed dry and the last measurement can be recorded as the dry soil weight.
   3. In the operating software, go to Measure Components and click on the Calculate Soil Dry Weight tab. Fill in the soil wet and dry weights for each sample, click Apply and Finish (Supplementary Figure 3).
2. Alternatively, manually calculate SWC using equation shown below.
   
3. Average the two SWC measurements taken manualy from at least three pots. Select the Measure Components tab and click on Calculate Soil Dry Weight the θg [g/g] value, click Apply and Finish. The soil dry weights of all of the experiment pots will be calculated automatically by the software (assuming that all of the pots in the experiment contain the same medium; Supplementary Figure 1D and Supplementary Figure 3).
4. Apply the irrigation treatments. Irrigation scenarios can be applied by composing a step-by-step treatment plan.
   1. To compose a new irrigation treatment plan, go to Irrigation Treatment, click on Create New, and name the new treatment. Open the specific treatment in the list of irrigation treatments and click the on the default “00:00”.
      NOTE: In the main window (Supplementary Figure 4A), “Time” indicates the time the valve will open (i.e., the beginning of the irrigation treatment). “Valve” is the valve to be opened (A or B, depending on the valve that is connected to the desired solution). “Command Type” indicates the type of data that will be used to determine when the valve will be closed:
      1. By Time – How many seconds the valve will be open.
      2. By Weight – The weight gain/water (in grams) to be added to the pot via irrigation.
      3. By Transpiration – Irrigation can be applied differentially to each pot based on the transpiration of each individual plant over the previous day. The user can decide what percentage of the previous day transpiration will be applied during irrigation. (Under the well-irrigated condition, it is suggested to give the plant more than 100%, in order to wash the soil and compensate for plant growth.) Drought-treated plants should be given less water, with exact volumes based on the desired drought stress rate.
      4. By Sensors – Irrigation can be applied according to a sensor reading, such as apparent dielectric permittivity (which can be used to determine the VWC). Select the sensor type, the desired parameter and the desired parameter value.
   2. All possibilities include a Time Out option that will close the tap even if the set conditions were not reached. Set the Time Out for a period longer than the set conditions.
   3. After defining the irrigation treatments for the experiment, open the desired experiment in the list of experiments, open Treatment Scenario, open default Plan and select the first step (Supplementary Figure 4B).
   4. In Treatment, choose an irrigation treatment from the list. Then, in Termination, choose the appropriate condition to stop the current step and move on to the next one.
   5. After selecting an irrigation scenario, open the experiment’s Plants table (Supplementary Figure 2) and input the “Treatment” and “Step” for each plant. “Treatment” is the name of the treatment scenario and “Step” is the event number within the treatment scenario.
5. Plan a drought treatment.
   1. Each individual plant has a unique transpiration rate based on its size and location in the greenhouse. To enable a standard drought treatment (i.e., similar drying rate for all pots during the treatment), plan a drought scenario and control it via the system’s feedback-irrigation tool (Supplementary Figure 5).

8. Analyze the data using data analysis software

1. Open the Data Analysis software (e.g., SPAC Analytics). Click on the top right corner to select Control system and the name of the experiment (Supplementary Figure 6A). In the column of the left side of the screen, select Experiments (Supplementary Figure 6B) and type the name of the experiment in the Name bar under the Search section. The name of the experiment will appear below the Search section, in the Experiments section (Supplementary Figure 6C). Click on the experiment to open the Info and Plants sections (Supplementary Figure 6D).
2. In the Info section, edit the WUE start and WUE end dates for a period of at least 3 (preferably more) days before the start of the drought treatment and then click Update. The WUE and the R² value for every pot will appear in the Plants section. Choose to exclude any scale with a negative WUE value or an R² value of less than 0.5 by clicking on the “eye” symbol under the Active column, which will then turn red. This will exclude the selected scale (plant) from all further calculations. The data can be exported by clicking on the Export Data button in the Plants section (Supplementary Figure 6D).
3. In the column on the left side of the scree, click on Analysis. Different subsections will then appear: Graph viewer, Histogram, T-test, ANOVA and Piecewise linear curve.
4. Click on Graph viewer. In the Filters section, set the dates for the experiment.
   1. Click on Labels (please see Step 6) to select the combination of experimental groups (genotype) and treatment(s). Automatically, all of the pots in the selected group will appear in the Plant subsection. In that subsection, deselect any pots (plants) by clicking on them. Up to two different parameters of choice can be selected at one time as the “Y1 parameter” and “Y2 parameter”. Finally, click on Show Graph (Supplementary Figure 5).
   2. A line graph of the values of the selected parameter will appear in the Graph Viewer window for each plant. Remove data from individual plants or add to the graph by clicking on their legend symbols on the right of the graph. In the top right corner, there are also options for exporting the data as a spreadsheet and for enlarging the Graph Viewer window to fill the full screen (this raw data download function is relevant to all other windows). More options to modify the graph will appear if the cursor is moved to the top right corner of the screen (Supplementary Figure 5).
5. The histogram module presents the distribution of a single trait in and between populations for a given time period. To use this module, click on Histogram.
   1. In the Filters section, set the date and time, parameter, labels and plants as explained in step 8.4.1. Select multiple labels (groups) by clicking on the + symbol. Finally, click on Show Graph (Supplementary Figure 7).
   2. The histogram will appear in the Histogram section, in which there is the option to change the “Bins” and “Date” at the top of the screen. In the top right corner, there are various options as described in step 8.4.2. In the Location Diagram section, the actual location of the plants on the experimental table and their respective trait values can be seen (Supplementary Figure 7).
6. Click on T-test. To statistically compare the means of any measured trait of two groups, enter the dates, labels, plants and parameters in the “T-test Parameters” section, as explained in step 8.4.1.
   1. Set the range of hours to calculate the average values of the data points within the time period of interest (the default is a continuous 24-h presentation). Finally, click on Show Graph (Supplementary Figure 8).
   2. Two windows will appear on the right side of the screen. The top one is the Graph Viewer section for all of the plants selected from both groups. Below that window is the T-test section, in which will appear the comparison of the two groups as the t-test of the physiological parameter selected. Levels of significance can be adjusted by changing the α-value in the top left corner of the T-test section. A red dot will appear under values that are significantly different. In the top right corner, view various options, as described in step 8.4.2 (Supplementary Figure 8).
7. Click on ANOVA. To statistically compare the means of any measured trait across more than two groups, enter the dates, labels, plants and parameters in the “Filters” section, as explained in step 8.5.1.
   1. Select multiple labels (groups) by clicking on the + symbol (as in step 8.5). Set the range of hours. Finally, click on “SHOW GRAPH” (Supplementary Figure 9).
   2. In the ANOVA section, use an ANOVA test (Tukey’s HSD) to compare the physiological parameters of the different groups. Bars represent the standard errors (±SE). In the top right corner of the screen, there are various options as described in step 8.4.2. Click on the line graph to view a bar-graph comparison for a particular day. Different letters indicate groups that are significantly different from one another (Supplementary Figure 9A).
8. Presenting the relationship between whole-plant transpiration kinetics or stomatal conductance and VWC is a more accurate way to compare the physiological responses of different plants to drought, as compared to a time-based approach. Present this relationship using the “Piece-wise Linear Curve” function.
   1. Click Piecewise linear curve. Enter the dates, labels, plants and parameters (both the x-axis and the y-axis) and then set the range of hours in the “Filters” section, as explained above.
      NOTE: The “from” date should be as close as possible to the treatment start date.
   2. Set the x-axis parameter to be VWC and the y-axis parameter as the physiological parameter of choice (e.g., transpiration rate, stomatal conductance, etc.). Finally, click on Show Graph. In the “Filter” section, click on Select all recommendations and then click on Show Graph (Supplementary Figure 10).
      NOTE: Other physiological parameters (e.g., normalized transpiration, transpiration rate, plant initial weight, stomatal conductance, root flux, etc.) and environmental parameters (e.g., temperature, relative humidity, etc.) are easily obtained via the SPAC software (e.g. Supplementary Figure 9C). For more information regarding the theoretical background of their calculations, please see Halperin et al. (2017).