High-Throughput Screening Identification of Chemical Compounds That Affect Cold-Regulated Gene Expression in Arabidopsis thaliana Using an Excised Single Leaf

Materials and reagents

Biological materials

1. Arabidopsis thaliana harboring COR15Apro::LUC construct

See General note 1.

Reagents

1. Murashige and Skoog plant salt mixture (MS salt) (Wako, catalog number: 392-00591)

2. Sucrose (Wako, catalog number: 196-00015)

3. 2-Morpholinoethanesulfonic acid monohydrate (MES) (Dojindo, catalog number: 343-01621)

4. D-Luciferin, potassium salt (GOLDBIO, catalog number: LUCK-1G)

5. Cycloheximide (Wako, catalog number: 037-20991)

6. Triton^TM X-100 (ICN Biomedicals Inc., catalog number: 807426)

7. Dimethyl sulfoxide (DMSO) (Wako, catalog number: 043-07216)

8. Ethanol (Wako, catalog number: 057-00456)

9. Chlorine-based bleach (e.g., KAO Kitchen Haiter)

10. Autoclaved dH₂O

Solutions

1. 0.5× MS liquid medium (see Recipes)

2. 50 mM D-Luciferin (see Recipes)

3. 2 mg/mL cycloheximide (see Recipes)

4. 20% (w/v) Triton X-100 (see Recipes)

5. MS-Luciferin solution (see Recipes)

6. 70% (v/v) ethanol (see Recipes)

7. Seed sterilization solution (see Recipes)

8. 0.1% agar solution (see Recipes)

Recipes

1. 0.5× MS liquid medium

Reagent	Final concentration	Quantity or Volume
MS salt	0.5×	0.23 g
MES	0.5 g/L	0.05 g
Sucrose	1%	1 g
dH2O		100 mL

This recipe is for two flasks. Adjust the volume to the amount you need. In general, divide the 100 mL of 0.5× MS liquid medium into two flasks (50 mL each) and then autoclave the flasks wrapped with aluminum foil. Autoclaved medium can be stored for at least two weeks at room temperature and under sterile conditions.

2. 50 mM luciferin

Reagent	Final concentration	Quantity or Volume
D-Luciferin, potassium salt	50 mM	0.2 g
dH2O		12.6 mL

Smaller amounts can be prepared depending on the objective. Luciferin (50 mM) can be aliquoted into 1.5 mL tubes and stored at -80 °C.

3. 2 mg/mL cycloheximide

Reagent	Final concentration	Quantity or Volume
Cycloheximide	2 mg/mL	2.4 mg
DMSO		1.2 mL

In this study, 2 mg/mL of cycloheximide was used because the concentration of the chemical compounds in our library was 2 mg/mL [7]. This concentration can be modified depending on the assay.

4. 20% (w/v) Triton X-100

Reagent	Final concentration	Quantity or Volume
TritonTM X-100	20% (w/v)	20 g
dH2O		100 mL

This recipe is for non-sterile conditions. You may use a commercially available sterile-filtered Triton X-100 if necessary.

5. MS-Luciferin solution (for 100 wells)

Reagent	Final concentration	Quantity or Volume
0.5× MS liquid medium	0.5×	20 mL
50 mM luciferin	0.5 mM	200 μL
0.1% (w/v) Triton X-100	0.001%	200 μL

Dilute 20% (w/v) Triton X-100 in dH₂O.

6. 70% (v/v) ethanol

Reagent	Final concentration	Quantity or Volume
Ethanol	70% (v/v)	7 mL
dH2O		Up to 10 mL

7. Seed sterilization solution

Reagent	Final concentration	Quantity or Volume
Chlorine-based bleach	30%	3 mL
20% (w/v) Triton X-100	0.02%	10 μL
dH2O		Up to 10 mL

8. 0.1% agar solution

Add 0.1 g of agar for plant culture in 100 mL of dH₂O and autoclave.

Laboratory supplies

1. 96-well PCR plate (e.g., BIO-BIK, catalog number: 3425-00)

2. 96-well culture plate (e.g., Nunc 96-well flat bottom, catalog number: 136101)

3. Stainless steel scissors

4. Stainless steel tweezers

5. Kimwipe and Kimtowel (Nippon Paper Cresia); these should be autoclaved if the experiments require sterile conditions

6. 100 mL flask (e.g., Iwaki, catalog number: 4980FK100)

7. Microtube 1.5 mL (e.g., SSIbio, catalog number: 1210-00)

8. 15 mL and 50 mL conical tubes (e.g., Falcon)

Equipment

1. Varioskan Flash microplate reader (Thermo Fisher Scientific)

2. LuminoGraph I imaging device (ATTO Co. Ltd., Japan) (Optional)

3. Growth chamber (e.g., Nippon Medical & Chemical Instruments Co., Ltd.)

4. Multichannel pipette

Software and datasets

1. SkanIt Software 2.4.3 (operating software for Varioskan Flash microplate reader)

2. ImageSaver 6 (operating software for LuminoGraph I chemiluminescence imaging device) (Optional)

3. CS Analyzer image analysis software (attached to LuminoGraph I) (Optional)

Procedure

A. Submerged culture of Arabidopsis plants in MS liquid medium

1. Sterilize Arabidopsis seeds. Place Arabidopsis seeds into a 1.5 mL tube, add 1 mL of 70% ethanol to the tube, and mix by rotation for 5 min. Remove 70% ethanol using a pipette, add 1 mL of seed sterilization solution (see Recipes) to the tube, and mix by rotation for 10 min. Remove the sterilization solution using a pipette and perform five washing cycles with autoclaved dH₂O. Suspend seeds in 0.1% agar solution.

2. Place 20–25 seeds into each 100 mL flask containing 50 mL of 0.5× MS liquid medium using a pipette.

3. Keep the flask at 4 °C for 2–3 days to synchronize seed germination.

4. Incubate the flask at 22 °C under continuous light (60–80 μE) in a growth chamber to grow plants in a submerged culture. Generally, it takes 11–12 days to obtain plants suitable for our assay (Figure 1).

Figure 1. Submerged Arabidopsis thaliana culture in a 100 mL flask

B. Preparation of compounds in a 96-well PCR plate

1. Dispense 2 μL of chemical compounds of your interest into each well of 96-well PCR plates. In this study, we used compounds dissolved in DMSO. This preparation can be performed on the day before the assay. The plate can be stored at -25 °C (or -80 °C for long-term storage).

Critical: Negative and positive controls should be included in the plate. Use four wells each for DMSO (negative control) and cycloheximide (positive control). Use column number 12 for the controls. You may increase the number of controls if necessary. See General note 2.

C. Preparation of a 96-well culture plate containing MS liquid medium and luciferin

1. Dispense 204 µL of MS-Luciferin solution (see Recipes) into each well of a 96-well culture plate.

D. Excise a single leaf from cultured Arabidopsis plants and place it into a culture plate

1. Excise true leaves from cultured Arabidopsis thaliana and place a single leaf into each well. In general, transfer the cultured Arabidopsis from a flask to a plastic container (Figure 2). After removing excess MS medium using the Kimwipe, cut true leaves and put them into each well using tweezers. Alternatively, you may first cut the leaves in a plastic container filled with MS medium and then remove the excess MS later. This procedure can be performed under sterile conditions if necessary. In this case, the plastic container and Kimwipe must be autoclaved before use. See General note 3.

Figure 2. Overview of the preparation of a 96-well culture plate containing a single leaf excised from Arabidopsis. This procedure can be performed under sterile conditions if necessary.

E. Incubation of each single leaf with compounds

1. Take 2 μL of each compound from a 96-well PCR plate and put it into a 96-well culture plate (Figure 3). Use a multichannel pipette to reduce the operation time.

2. Shake the culture plate briefly.

3. Incubate the plate at 4 °C under continuous light for 24 h (Figure 3). In this case, induce COR15A pro::LUC strongly at low temperatures after 24 h. Incubation conditions can be altered depending on the objective.

Figure 3. Overview of the incubation of each single leaf with the compound

F. Measurement of luciferase activity using Varioskan Flash microplate reader

1. After incubation for an appropriate time, transfer the plates to a microplate reader.

2. Record chemiluminescence:

a. Start SkanIt software.

b. Measure luciferase activity using luminometric measurement mode.

3. Once the measurement is performed, export the data as a text file (.txt).

G. Measurement of luciferase activity using LuminoGraph I

Optional. See General note 4.

1. After incubation for an appropriate time, place the plate into a LuminoGraph I (or another chemiluminescence imaging device).

2. Capture chemiluminescence:

a. Start ImageSaver 6.

b. Use Sum (constant-time) mode with high sensitivity (8×8) to capture the images. We usually use the sum of the 1-min exposure.

3. Quantify luminescence in each well using CS Analyzer software (or other appropriate software).

See General notes 5 and 6.

Data analysis

Export the raw values (luminescence units) to Microsoft Excel and open the data. Calculate the average luminescence units of the positive (CHX) and negative (DMSO) controls and then express the relative luminescence of each well as a percentage of the negative control (Figure 4). Screen for compounds that caused at least 90% inhibition of luciferase activity after low-temperature treatment in the first screening (Figure 4). You can combine percentile data of multiple plates into a single figure, as we did in our previous study [7].

Figure 4. Summary of data analysis. Raw data should be converted to a percentile of negative control (DMSO) to identify compounds that inhibit cold-induction of COR15Apro::LUC. A chemiluminescence imaging device can be used as an alternative to a microplate reader (Figure 5). You need to quantify the luminescence from each well using image analysis software.

Figure 5. Chemiluminescence image of the 96-well culture plate containing a single leaf of COR15Apro::LUC Arabidopsis after cold treatment for 24 h. Dark wells contain inhibitors of cold-induction of COR15Apro::LUC identified in the previous study [7].

Validation of protocol

This protocol or part of it has been used and validated in the following research article:

1. Kitawaki et al. [7] Chemical screening approach using single leaves identifies compounds that affect cold signaling in Arabidopsis. Plant Physiol.

General notes and troubleshooting

1. Choice of biological materials: We used Arabidopsis thaliana harboring COR15Apro::LUC construct to screen chemical compounds that affect cold-regulated gene expression. However, this material can be changed, depending on the objective.

2. Sample size of control: Positive and negative controls are the most important factors that affect data interpretation. Four positive and negative controls were added to each plate. However, owing to the nature of the assay, there was considerable variation in the luciferase activity between the leaves. Therefore, you may increase the number of control samples depending on the variation in the assay.

3. Effects of leaf size on luciferase activity: We previously reported the effect of leaf size on the luciferase activity of COR15Apro::LUC [7]. We confirmed that the differences in size did not significantly affect the low-temperature responses.

4. Choice of measurement device: If available in your institution, a microplate reader should be your first choice for detecting luciferase activity. It directly quantifies the luciferase activity, which makes data processing easier. Alternatively, chemiluminescence imaging devices can be used for this purpose. However, in this case, the signal intensities need to be quantified using image analysis software.

5. Measurement of luciferase activity: If the luciferase activity is undetectable or too low, check the sensitivity of the device (e.g., use a high-sensitivity mode). The detection time may also be increased.

6. Replication of test samples: In a previous study [7], we used two independent leaves for each compound and calculated the average to evaluate the effects of each compound. If the volume of available compounds is limited, one leaf may be used for each compound during the first screening. However, we recommend increasing the number of leaves during the second screening period.

Acknowledgments

We thank Ms. Saori Hamada (University of Miyazaki, Japan) for her technical assistance. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (20H02917 and 25K01953 to Y.I.I., 24K01683 and 24K21843 to T.I.) and a Sasakawa Scientific Research Grant from the Japan Science Society (to K.K. and R.M.).

Competing interests

Authors declare that they have no competing interests.

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