Materials and reagents

Biological materials

1. Transgenic Arabidopsis thaliana seeds harboring the LUC gene under the control of NRT2.1 promoter [2]

1. Sodium hypochlorite (FUJIFILM Wako Pure Chemical, catalog number: 197-02206)

2. D-luciferin potassium salt (FUJIFILM Wako Pure Chemical, catalog number: 120-05114)

3. Potassium nitrate (FUJIFILM Wako Pure Chemical, catalog number: 160-04035)

4. Ammonium succinate (Kanto Chemical, catalog number: 01319-30)

5. Murashige and Skoog salts without nitrogen, phosphorus, and iron (FUJIFILM Wako Pure Chemical, special order)

6. 2-Morpholinoethanesulfonic acid (MES) (Dojindo, catalog number: 343-01626)

7. Potassium hydroxide (FUJIFILM Wako Pure Chemical, catalog number: 168-21815)

8. Potassium dihydrogen phosphate (FUJIFILM Wako Pure Chemical, catalog number: 169-02425)

9. Iron (II) sulfate heptahydrate (FUJIFILM Wako Pure Chemical, catalog number: 094-01082)

10. Sucrose (FUJIFILM Wako Pure Chemical, catalog number: 196-00015)

1. MES buffer (see Recipes)

2. 10 mM iron (II) sulfate solution (see Recipes)

3. 1 M potassium dihydrogen phosphate solution (see Recipes)

4. 100 mM ammonium succinate solution (see Recipes)

5. 1× Murashige and Skoog solution (N-, P-, Fe-free) (see Recipes)

6. 0.1× N-free MS medium (see Recipes)

7. 1 M potassium nitrate stock solution (see Recipes)

8. 10 mM D-luciferin potassium stock solution (see Recipes)

1. 170 mM MES buffer

   Note: Adjust the pH of 170 mM MES buffer to 5.7 using potassium hydroxide solution. Filter-sterilize the buffer and store at room temperature.

   Reagent	Final concentration	Quantity or Volume
   2-Morpholinoethanesulfonic acid	170 mM	3.6 g
   Potassium hydroxide	n/a	n/a
   Ultrapure water (Milli-Q)	n/a	Up to 100 mL
   Total	n/a	100 mL




2. 10 mM iron (II) sulfate stock solution (10 mL)

   Note: Filter the solution, aliquot into 1.5 mL tubes, and store at -20 °C to prevent precipitation.

   Reagent	Final concentration	Quantity or Volume
   Iron (II) sulfate heptahydrate	10 mM	27.8 mg
   Ultrapure water (Milli-Q)	n/a	Up to 10 mL
   Total	n/a	10 mL




3. 1 M potassium dihydrogen phosphate stock solution (10 mL)

   Note: Autoclave or filter-sterilize the solution and store at room temperature.

   Reagent	Final concentration	Quantity or Volume
   Potassium dihydrogen phosphate	1 M	1.36 g
   Ultrapure water (Milli-Q)	n/a	Up to 10 mL
   Total	n/a	10 mL




4. 100 mM ammonium succinate stock solution (10 mL)

   Note: Autoclave or filter-sterilize the solution and store at room temperature.

   Reagent	Final concentration	Quantity or Volume
   Ammonium succinate	100 mM	0.15 g
   Ultrapure water (Milli-Q)	n/a	Up to 10 mL
   Total	n/a	10 mL




5. 1× Murashige and Skoog medium (without N, P, and Fe) (1 L)

   Note: To conduct studies involving nutrient-deficiency treatments, make a specialized Murashige and Skoog plant salt mixture lacking KNO₃, NH₄NO₃, KH₂PO₄, and FeSO₄, and store the solution at 4 °C.

   Reagent	Final concentration	Quantity or Volume
   Murashige and Skoog plant salt mixture (N-, P-, and Fe-free)	1×	1 bag
   Ultrapure water (Milli-Q)	n/a	Up to 1 L
   Total	n/a	1 L




6. 0.1× N-free MS medium + 500 µM ammonium succinate (100 mL)

   Note: Mix all components, except iron (II) sulfate solution, and autoclave. After autoclaving, add the iron (II) sulfate solution and mix thoroughly. Store the solution at 4 °C.

   Reagent	Final concentration	Quantity or Volume
   1× Murashige and Skoog medium (without N, P, and Fe)	0.1×	10 mL
   Sucrose	0.5% (w/v)	0.5 g
   100 mM ammonium succinate stock solution	500 µM	0.5 mL
   1 M potassium dihydrogen phosphate stock solution	125 µM	12.5 µL
   170 mM MES stock solution	3.5 mM	2.1 mL
   10 mM iron (II) sulfate stock solution (add after autoclave)	10 µM	100 µL
   Ultrapure water (Milli-Q)	n/a	Up to 100 mL
   Total	n/a	100 mL




7. 1 M potassium nitrate stock solution (10 mL)

   Note: Autoclave or filter-sterilize the solution and store at room temperature.

   Reagent	Final concentration	Quantity or Volume
   Potassium nitrate	1 M	1.0 g
   Ultrapure water (Milli-Q)	n/a	Up to 10 mL
   Total	n/a	10 mL




8. 10 mM luciferin potassium stock solution (10 mL)

   Note: Aliquot the solution into 1.5 mL tubes and store at -80 °C.

   Reagent	Final concentration	Quantity or Volume
   Luciferin potassium	10 mM	31.8 mg
   Ultrapure water (Milli-Q)	n/a	Up to 10 mL
   Total	n/a	10 mL

Laboratory supplies

1. Black 96-well microplates (Greiner, F-bottom, catalog number: 655077)

2. Transparent lids for 96-well plates (e.g., TPP, catalog number: 92696)

3. 1.5 mL tubes (e.g., Watson, catalog number: 131-415C)

4. 200 µL pipette tips (e.g., Watson, catalog number: 110-705C)

5. Surgical tape (e.g., 3M Company, catalog number: 1530-0)

6. Beaker

Equipment

1. Microplate reader (Tecan, model: Infinite M1000)

2. Electronic balance

3. Autoclave

4. Tabletop centrifuge

5. pH meter

6. Magnetic stirrer

7. Clean bench

8. Growth chamber (equipped with LED lights)

9. Pipettes (200 µL, single- and 8-channel)

Software and datasets

1. Magellan software (Tecan)

2. Microsoft Excel (Microsoft)

Procedure