Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system

Summary Environmental stimuli evoke transient increases of the cytosolic Ca2+ level. To identify upstream components of Ca2+ signaling, we have optimized two forward genetic screening systems based on Ca2+ reporter aequorin. AEQsig6 and AEQub plants were used for generating ethyl methanesulfonate (EMS)-mutagenized libraries. The AEQsig6 EMS-mutagenized library was preferably used to screen the mutants with reduced Ca2+ signal response due to its high effectiveness, while the AEQub EMS-mutagenized library was used for screening of the mutants with altered Ca2+ signal response. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020) and Zhu et al. (2013).

considered a better choice than the AEQ35S EMS library because 35S promoter-driven apoaequorin is often silenced when crossed with T-DNA insertion mutant or transformed with 35S promoter-driven transgenes for gene functional analysis, most likely due to siRNAmediated transcriptional silencing (Mlotshwa et al., 2010). Therefore, we recommend an AE-Qub EMS library or a genetic background of interest expressing an AEQub construct for the screening of the mutants with altered Ca 2+ signal response. This protocol aims to screen mutants with altered Ca 2+ signals using Aequorin-based luminescence imaging. Screening Ca 2+ responsive mutants with the plate reader can be conducted by following two other protocols (Tanaka et al., 2013, Mittal et al., 2020.
CRITICAL: EMS is extremely toxic. Handle EMS with gloves in a chemical fume hood. Leave EMS contaminated water and labware in a beaker containing 1 mol/L NaOH for at least one week in the hood with warning labels for decontamination.
Note: To avoid poor seed vigor or growth defect of plant materials, the EMS mutagenized pool should be generated freshly from the plants under non-stress conditions. The M1 seedlings are grown under an isolative space to avoid cross-pollination.

MATERIALS AND EQUIPMENT
A cooled charge-coupled device (CCD, Princeton Instruments, New Jersey, USA) controlled by Win-View/32 (Roper) is used to acquire Ca 2+ luminescence images. The optimal working temperature for the CCD camera is À100 C. The CCD imaging system should be placed in a dark room to avoid the interference of light. The equivalent imaging system can be used for the same purpose. Also, a luminometer (GLOMAX, Promega) or Microplate Reader (SpectraMax i3x, Molecular Devices) with or without an auto-pump injector system can be used for quantitative measurement of Ca 2+ signal dynamics.
CRITICAL: EMS is extremely toxic. Handle EMS with gloves in a chemical fume hood. Leave EMS contaminated water and labware in a beaker containing 1 mol/L NaOH for at least one week in the hood with warning labels for decontamination. Adjust pH to 5.7 with 0.5 M KOH, and autoclave at 121 C for at least 15 min. Cool to $60 C and pour into disposable Petri dish after autoclave. Store MS plates at 4 C-8 C for up to 3 months.
Adjust pH to 5.7 with 0.5 M KOH, and autoclave at 121 C for at least 15 min. Cool to $60 C and pour into disposable Petri dish after autoclave. Store MS plates at 4 C-8 C for up to 3 months.
*Note: After autoclave and cooled to $60 C, add cefotaxime stock solution and mix thoroughly, then pour into disposable Petri dish. Store MS plates at 4 C-8 C for up to 3 months.
Alternatives: Phytagel (3 g) could be changed to agar (7 g) for 1/2 MS horizontal plates with 0.7% agar. The purpose of these steps is to prepare plant materials for analyzing Ca 2+ signal response.
CRITICAL: Excessive light in the growth chamber and excessive sucrose in growth media should be avoided because the stressed seedlings are not suitable for monitoring Ca 2+ signal response. Coelenterazines are very sensitive to oxidation and are used as a chemiluminescent indicator of ROS accumulation (Dubuisson et al., 2000). To avoid resulting problems, seedlings must be grown under optimal growth conditions, and aequorin should be reconstituted in the dark. We recommend screening 10-20 plates per day for skilled researchers.

Analyzing transient Ca 2+ signal response
Timing: 6-9 h The purpose of these steps is to monitor the Ca 2+ signal response of M2 AEQsig6 seedlings for screening mutants.
7. Draw a big arrowhead on the back of the plate to orientate the plate. 8. Spray the 9-day-old seedlings evenly with $2-3 mL/per dish of coelenterazine working solution (NanoLight, final concentration 10 mM in sterile dH 2 O, 0.1% Triton X-100 with a laryngeal or nasal spray).
CRITICAL: Apoaequorin binds to a prosthetic group, coelenterazine, to be converted to aequorin that reacts with Ca 2+ (Shimomura et al., 1962). In the presence of Ca 2+ , coelenterazine is decomposed into coelenteramide and carbon dioxide with the emission of blue light at approximately 465 nm (Mithofer and Mazars, 2002). Coelenterazines are stable in solid form when stored at À80 C under nitrogen or argon, but unstable under light or in an aqueous solution because they are prone to be oxidized. The stock solution (2 mM in ethanol) should be prepared in ethanol or methanol and stored at À80 C in light-tight tubes and handled in the dark. Do not use DMF or DMSO to prepare stock solution because these solvents cause oxidation of coelenterazine. Use freshly prepared working solution.
9. Incubate Petri dishes for 4-6 h at room temperature (20 C-25 C) in the dark for aequorin reconstitution.

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10. Pre-cool down the operating temperature of the CCD camera to À100 C. 11. Transfer the Petri dish to the light-tight box of the CCD imaging apparatus in the dark.
CRITICAL: To prevent chlorophyll autofluorescence from interfering with the luminescence recording, do not expose the seedlings to light after applied coelenterazine. If the seedlings are occasionally exposed, keep in the dark for 5 min to minimize chlorophyll autofluorescence.
12. Treat the seedlings with 600 mM mannitol in water (30 mL per 150 mm 3 15 mm Petri dish) in the dark. Note: The solution of given stimuli should submerge the seedlings, but not too much.
13. Acquire the luminescence image (Image1) continuously for 2-5 min, immediately after treatment. 14. Discard the mannitol solution. 15. Place the Petri dish back to the light-tight box of the CCD imaging apparatus as previously orientated. 16. Treat the seedlings with 10 mM H 2 O 2 in water (30 mL per 150 mm 3 15 mm Petri dish). 17. Acquire the luminescence image (Image2) continuously for 2-3 min immediately after treatment. 18. Compare the Image1 with the Image2. 19. Pick up seedlings showed weaker luminescence in Image1 and normal luminescence in Image2 (about 0.5% of the total seedlings).
Note: We assumed that most mutations do not affect the Ca 2+ signal. Thus the majority of the M2 seedlings are considered as wild-type controls.
20. Rinse the mutant candidates with 25 mg/L cefotaxime solution in sterile-deionized water 2 times.
Note: The use of cefotaxime reduces contamination by micro-organisms (Mittal et al., 2020).
21. Transfer the mutant candidates to 1/2 MS medium (without sucrose) with 25 mg/L cefotaxime under low light for recovery.
Note: Dissolve cefotaxime in 1/2 MS medium. This step is not necessary for stimuli that do not cause severe damage.
22. Transfer the mutant candidates to the soil after 1-2 days recovery, and grow seedlings under optimal growth conditions. CRITICAL: Steps 14-18 are optimal and should be carefully designed. Although these steps reduce the false positive mutant candidates, they may also have mutant candidates escaped. Some stimuli may share similar receptors or upstream regulators (Laohavisit et al., 2020, Wu et al., 2020. The alternative strategy is to pick up about 1.5%-2% of the total seedlings only according to Image1 ( Figure 1A). The recommended ranges for stimuli were listed in Table 2.
Pause point: Seeds can be safely stored at dry conditions at room temperature (20 C-25 C) for 2-5 years. As an alternative, seeds can be safely stored at À20 C for more than 5 years with high viability. The purpose of these steps is to confirm the defective Ca 2+ signal response of these mutant candidates isolated during the first-round screen.

Second-to fourth-round screens
23. Sterilize the seeds of mutant candidates and the wild type AEQsig6, as steps 1 and 2. 24. Plant seeds evenly on a 90 mm 3 15 mm round or 100 mm 3 15 mm square disposable Petri dish with a 0.9-1 cm interval on 0.3% Phytagel containing 1/2 MS nutrients, 1% sucrose, pH5.7 (at least three plates for each line). Prepare plant materials as steps 4-6. 25. Treat the 9-day-old seedlings with 600 mM mannitol, 10 mM H 2 O 2 or other stimuli, as steps 7-13. 26. Acquire the luminescence image (Image3) continuously for 2-5 min, immediately after treatments. 27. Discard the mannitol solution and place the Petri dish back to the light-tight box of the CCD, as steps 14 and 15. 28. Treat the seedlings with discharging buffer (2 M CaCl 2 in 20% ethanol).
Note: The high concentration of ethanol destroys the cell membrane, allowing Ca 2+ to penetrate the cells and discharge the remaining aequorin.
29. Acquire the luminescence image (Image4) continuously for 5 min, immediately after treatments. 30. Mutant candidates with reduced luminescence in Image3, and standard or enhanced luminescence in Image4 are preferable for third-to fourth-round screens ( Figure 1B). 31. Select candidate mutants with reduced Ca 2+ signal for third-to fourth-round screens and transfer the mutant plant to soil and harvest seeds from 8 individual seedlings using the single pedigree-based seed collection to obtain the homozygous, in case of a dominant mutation. 32. Analyze phenotypes of mutant candidates under osmotic stress. It should be aware that stimuli responses are controlled both by Ca 2+ -dependent and -independent processes (Kadota et al., 2014, Chen et al., 2020, Yuan et al., 2014. (C) A false mutant candidate with reduced osmotic stress-induced Ca 2+ luminescence response (left), but with reduced discharged Ca 2+ luminescence intensity (right). Scale bars, 1 cm.

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Screening AEQub EMS library for altered Ca 2+ signal responsive mutants using the film adhesive seedling (FAS) system Preparing plant materials for the first-round screen Timing: 12 days The purpose of these steps is to monitor the Ca 2+ signal of M2 AEQub seedlings.
33. Prepare M2 AEQub seedlings for luminescence imaging. Sterilize EMS-mutagenized M2 seeds of AEQub plants with a 10% bleach solution containing 0.01% Triton X-100 or 0.1% HgCl 2 for 5 min and rinse with sterile water 4-5 times. Sow the sterilized seeds on a square plate (10 3 10 cm square Petri dish with grid) containing 1/2 MS, 1% sucrose, and 1.2% agar. Place plates vertically in a growth chamber for 7 days, after stratification at 4 C for 2 days.
34. Transfer the seedlings onto a film. Place an adhesive film (Thermo Scientific Adhesive PCR Plate Seals) on the top of 7-day-old seedlings growing on the vertical plate. Gently push the film by hand to ensure that seedlings adhere to the film, and peel the film gently to let the seedlings adhered to the film (Figures 2A-2C). 35. Incubate the seedlings with coelenterazine. Place the adhered seedlings onto the square plate (10 3 10 cm) containing 15 mL of 1 mg/mL h-coelenterazine (15 ml of 1 mg/mL coelenterazine added to 15 mL dH 2 O). Incubate the seedlings at room temperature (20 C-25 C) for 4 h to overnight (9-11 h) ( Figures 2D and 2E). 36. Prepare for luminescence imaging. Discard the h-coelenterazine solution and place the film with seedlings face-up within the same plate. Leave the plate in the dark for 5 min ( Figure 2F). 37. Acquire luminescence images. In the dark, place the plate on the stage of the CCD imaging system. Acquire images immediately and continuously for 2-5 min upon adding 20 mL of stimuli solution to the plates ( Figures 2G and 2H). The concentrations of stimuli were listed in Table 2. 38. Analyze luminescence images and pickup mutant candidates. Set the same display range for all acquired luminescence images. Pick the seedling with altered Ca 2+ luminescence, let them recover on 1/2 MS medium (without sucrose) with 25 mg/L cefotaxime for two days before transferring to soil.

Second-to fourth-round screens
Timing: 2 weeks The purpose of these steps is to confirm the mutant candidates isolated during the first-round screen.
39. Sterilize the seeds of mutant candidates and their wild-type AEQub and grow the seedlings on a square plate as indicated in Figures 3A-3C. Prepare four replicates of the same orientated seedlings for FAS imaging as steps 33-36. 40. Acquire images at an interval of 40 s for 160 s immediately upon adding 20 mL of 100 mM NaCl, 400 mM mannitol, 1 mM H 2 O 2 to each plate, respectively ( Figures 3D-3F). Acquire discharged images by applying 20 mL of discharge solution (2 M CaCl 2 in 20% ethanol) to the plate containing the previous stimuli-treated seedlings ( Figure 3G). 41. Analyze luminescence images by comparing Ca 2+ signal response images to its discharge images of mutant candidates. Mutant candidates with enhanced or reduced Ca 2+ luminescence

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to given stimuli, but with similar luminescence intensity of discharged images should be considered for further verification by performing third-to fourth-round confirmation ( Figure 3H).
Note: As noted above, the physiological status of seedling significantly affects Ca 2+ signal response. To avoid the interference by defect of seed germination and growth, we recommend using the different batches of mutant seeds together with their wild-type AEQsig6 or AEQub that were harvested under the same growth condition for each round screen.
CRITICAL: Mutant candidates with reduced luminescence intensity of discharged aequorin should be eliminated ( Figure 1C and mutant 11 in Figures 3D-3G). These could be caused by reduced expression of the apoaequorin gene due to mutations or silencing, or defects in coelenterazine absorption or aequorin reconstitution as described above.
Quantitative Ca 2+ measurement using a luminometer with AEQ seedlings Timing: 10 h The purpose of these steps is to determine the Ca 2+ signal dynamical features of mutants in response to given stimuli.
42. Place a single 7-day-old AEQsig6 or AEQub seedling into a 1.5 mL Eppendorf tube that contains 1 mL of 1 mg/mL h-coelenterazine and place them in a light-seal box and leave it on a shaker (80 rpm/min) at room temperature overnight (20 C-25 C, 9-11 h). 43. Place the tube contains the seedling into the luminometer chamber. Acquire the signal for 60 s at 1 s intervals for resting luminescence. 44. Acquire luminescence intensity (L stimuli ) at 1 s intervals for 80-120 s (depended on the duration of Ca 2+ luminescence response) immediately after adding 1 mL of 400 mM mannitol, or 100 mM NaCl, or 5 mM H 2 O 2 solution into the tube. 45. Discard the stimuli solution, and acquire the luminescence intensity (L rest ) at 1 s intervals about 2 min until values were within 5% of the highest discharged value, immediately after adding 1 mL of discharging buffer (2 M CaCl 2 in 20% ethanol).

Note:
Steps 42-45 can be adjusted depending on the type of luminometer used (Mittal et al., 2020, Tanaka et al., 2013. 46. The total luminescence (L max ) is calculated by L rest plus L stimuli . The calibration equation is adopted from the previously described equation : pCa = 0.332588 (-logk) + 5.5593, where k is a rate constant equal to stimuli triggered aequorin luminescence (L stimuli ) divided by total luminescence (L max ). Mutants with either altered amplitudes or durations are shown in Figure 4.

Whole-genome resequencing
Timing: 10 months The purpose of these steps is to clone the gene with the mutation responsible for the altered Ca 2+ signal response.
47. Backcross the mutant with wild-type AEQsig6 or AEQub, and analyze the mannitol-induced Ca 2+ signal in F1 seedlings. When F1 shows a wild-type phenotype, it is a recessive mutation, and vice versa, it is a dominant mutation. We suppose the mutation is recessive here. 48. Generate the backcrossed F2 population, the ratio of mutant-like and WT-like seedlings is 1:3 in the backcrossed F2 population.  (Abe et al., 2012). 53. Resequence the mutant parent line and the wild-type AEQsig6. 54. Look for SNP changes only present in the mutant bulk as described previously (Abe et al., 2012).
Alternatives: Map-based cloning using Wassilewskija (Ws) background also works (Yuan et al., 2014), but will also require a larger population to be screened.

EXPECTED OUTCOMES
The mutants with reduced or enhanced Ca 2+ signal response to osmotic stress or given stimuli as described could be isolated. These two systems are designed to identify receptors, sensors, or upstream regulators in signaling pathways that involve Ca 2+ response. We screened about 100,000 M2 seedlings, and about 567 mutant candidates were isolated from the initial screen using the AEQsig6 EMS mutagenized library. Several mutants were selected for further analysis after second-to fourthround screens, together with mutant growth phenotype analysis under osmotic or other abiotic stress treatments.

QUANTIFICATION AND STATISTICAL ANALYSIS
All luminescence imaging data are captured and analyzed by Winview/32. We adjust an appropriate range of display layouts, which allows the visualization of luminescence signals from the majority of seedlings. Subtraction of the background signal from the raw signal resulted in the Ca 2+ luminescence signal of individual seedlings.
Quantitative Ca 2+ measurement using a luminometer is described in step 46. The detailed data processing is provided in Table S1, using the raw data for Figure 4B.

LIMITATIONS
Although the aequorin-based Ca 2+ luminescence system provides a viable platform to identify genes of interest in Ca 2+ response-related signaling pathways, it still has the following limitations. 1) Plants display tissue-and stimuli-specific Ca 2+ signal responses as described previously (Zhu et al., 2013). For example, H 2 O 2 , NaCl, cold and amino acids cause more substantial and robust Ca 2+ luminescence in leaves. In contrast, mannitol-induced Ca 2+ responses are relatively weaker in leaves, and somewhat more potent in roots. Tissue-specific Ca 2+ signal responses make the isolation of osmotic responsive mutants more challenging and less efficient using the first protocol, but more accessible using the second protocol. Even performed by skilled researchers, achieving a more robust and reproducible Ca 2+ luminescence response is still challenging.
2) Screening and cloning are labor-intensive and time-consuming and require researchers with welltrained skills, stable funding, expensive equipment, and enough space. Detailed experiments should be well designed in advance.

Potential solution
It is recommended to optimize the strength of stimuli for triggering Ca 2+ luminescence response to screen either reduced or enhanced Ca 2+ responsive mutants. Generally, for a screening of reduced Ca 2+ responsive mutants, the use of high strength stimuli should be considered. In contrast, for a screening of enhanced Ca 2+ responsive mutants, the low strength of stimuli should be applied. The weak Ca 2+ luminescence response could be caused: 1) the suboptimal concentration and application method of stimuli; 2) the suboptimal concentration and co-incubation condition of coelenterazine; 3) the oxidation of coelenterazine that makes screening less effective; 4) the seedlings are too young or too old, or suffer from stress before imaged; 5) the CCD camera system is not on optimal work conditions. Therefore, the strength of any given stimuli should be optimized to meet the purpose of screening. The concentration of coelenterazine and stimuli mentioned in the protocol should be adjusted based on the intensity of luminescence and increased or decreased if luminescence response is too weak or too strong.

Potential solution
It is recommended that EMS seeds are sieved through 0.25 mm mesh before planting on the plate. AEQsig6 seeds are planted evenly on a 150 mm 3 15 mm round disposable Petri dish with a 0.9-1 cm interval with about 200 seeds per dish (Figure 1). AEQub seeds are planted as 5 lines along the grid on a 10 3 10 cm square Petri dish with $200 seeds per dish (Figure 2). Grow plates on the optimal conditions to have uniform seedlings grown well on the plate and adhered to the film. FAS system allows a co-incubation of uniform seedlings adhered to the same film with coelenterazine to avoid the variations due to uneven spraying of coelenterazine. Therefore, the FAS system offers a more sensitive, reproducible, and reliable platform for monitoring Ca 2+ luminescence response as the advised performances were followed.

Potential solution
The optimal working temperature for the CCD camera is À100 C. Inappropriate working temperature leads to the strips-like noise signal on the full image. The CCD imaging system should be placed in a dark room to avoid visible light signal interference, which can cause dots-like noise.