AT7 protoplast system

2.12.1. Protoplasts preparations from AT7 cells

Arabidopsis 5 days-old AT7 cells were harvested by centrifugation at 800g for 5 minutes at room temperature. Cells were washed with 40 ml of 240 mM CaCl2

solution and centrifuged as described above. The supernatant was discarded and cells were resuspended in 60 ml cellulase solution. The cell suspension was divided into two Petri’s dishes and incubated for 20 hours at 26oC in the dark shaking at20 rpm. Before harvesting, the protoplasts were shaking 40 rpm for 20 minutes. They were transferred to a 50ml tube and centrifuged at 800g for 6 minutes at room temperature. The supernatant was discarded and protoplasts were resuspended in 25 ml of 240 mM CaCl2 solution and centrifuged at 800g for 6 minutes at room temperature. The protoplast pellet was resuspended with B5- sucrose solution and centrifuged at 800g for 6 minutes at room temperature. After this centrifugation step, the living protoplasts were floating in the solution, whereas dead protoplasts were positioned in the bottom of the tube. Floating protoplasts, which were ready for transfection, were collected to a new tube (modified from Dangl et al., 1987).

B5-sucrose solution Celullase solution

3.2 g Gamborg’s B5 medium (Sigma) for 1 liter 0.7g Cellulase (1.2 U/mg) 1 mg 2,4-Dichlorophenoxyacetic acid pH 7.50. 1625 g Mazerase (0.55 U/mg) 0.4 M Sucrose 60 ml 240 mM CaCl2

pH 5.7 adjusted with 0.1 M KOH Filter sterilized

2.12.2. Transfection of AT7 protoplasts

Plasmid DNA was transfected to protoplasts mediated by polyethylene glycol (PEG). In a 10 ml centrifuge tube, 200 μl of protoplast were mixed with 25μg of plasmid DNA (10 μg of a promoter construct and 5 μg of a standardization construct pBT10-UBIpro:LUC and 10μ g of a promoterless luciferase construct pBT10-LUC). To this mixture, 200 μl of PEG solution were added and incubated 15 minutes at room temperature. The incubation was stopped by adding 5 ml of 275 mM Ca(NO3)2 solution (pH 6.0) and protoplasts were centrifuged at 400g for 8 minutes at room temperature. The supernatant was discarded and protoplasts were resuspended in 7ml of B5-sucrose solution. The protoplasts were incubated at 26oC in the dark for 20 hours. For transient expression of miRNA precursors, protoplasts were transfected with 12.5 μg of a miRNA precursor construct and 12.5 μg of the pBT10 empty vector. For transient expression of miRNA precursor

and its putative target, 12.5 μg of each construct were transfected to protoplast (modified from Krens et al., 1982; Hain et al., 1985; Lipphardt et al., 1988).

PEG solution

25% PEG6000

100mM Ca(NO3)2

450mM Manitol

2.12.3. Harvesting protoplast

On the day following the transfection, protoplasts were mixed with 20 ml of 240mM CaCl2 solution and centrifuged at 400rpm for 10 minutes at 4oC. The supernatant was removed with the help of a vacuum pump until 1 ml was left. Protoplasts were resuspended and transferred to a 1.5 ml tube. After a brief centrifugation, 13000rpm for 10 seconds, the supernatant was removed and the protoplasts were frozen in liquid N2. Protoplasts were kept at -80oC until the protein or RNA extraction.

2.12.4. Protein extraction of protoplast

Measurement of promoter activity was done at the protein level. To this aim, protein extracts were prepared from transfected protoplasts. To each tube containing protoplast pellet, 800 μl of luciferase extraction buffer were added and tubes were shaken for 30 seconds. Protoplast debris was separated by centrifugation (10minutes at 4°C at 12000g) and the supernatant was transferred to a new 1.5 ml tube. The protein extract was kept on ice until the measurement of protein concentration, luciferase activity and GUS activity.

Luciferase extraction buffer

100 mM KH2PO4, pH 7,5

2.12.5. Protein quantification with Bradford

Protein quantification was done by Bradford assay (Bradford, 1976). For the standard curve, freshly prepared bovine serum albumin (BSA) dilutions of 1 μg, 2 μg, 5 μg, 10 μg, 15 μg and 20 μg per μl in Luciferase extraction buffer were used. Protoplast protein extract was diluted to 1:5 in the same buffer. For measurement, 800µl of diluted protein sample was added to 200µl of Protein Assay Dye Reagent (Bio-Rad) and incubated at room temperature for 20 minutes. After incubation time, the protein concentration was measured in a Biophotometer (Eppendorf) at 595nm wavelength. Comparison to a standard curve provided a relative measurement of protein concentration.

2.12.6. Luciferase Assay

For luciferase assay (Wood, 1991), 10 μl of protein extract were transferred to a glass tube and 100 μl of luciferase solution were added. The tube was briefly mixed and the measurement was immediately performed in a luminometer (MiniLum, BioScan, Washington DC, USA). Such measurement provides the relative light units (RLU) of the sample. The RLU value of the sample refers to the luciferase activitity by protein amount (μg) by second (RLU μg-1 _sec-1_).

Luciferase reaction buffer

20 mM Tricine 2.67 mM MgSO4 0.1 mM EDTA 33.3 mM DTT 270 μM CoA 470 μM D-Luciferin 530 μM ATP

2.12.7. GUS activity

Beta-glucoronidase fluorimetrical assay (Jefferson et al., 1986) is based upon the conversion of 4-methylumbelliferyl-beta-D-glucuronide (4MUG) into the fluorescent product 4-methylumbelliferyl (4MU) by beta-glucoronidase. To measure the GUS activity in protein extracts, 100 μl of protein extract were mixed with 100 μl of GUS-

solution in a MicroWell BlackTM _{plate. Measurement was made in a fluorimeter} (Fluoristar Optima, BMG LABTECH, Offenburg, Germany). The micro plate was incubated at 37oC and fluorescence was measured with excitation at 365 nm and emmision at 460 nm at three time points: 20, 40 and 60 minutes. The fluorimeter was calibrated with freshly prepared 4MU standard at different concentrations, ranging from zero to 4000 pMol of 4MU. The difference in fluorescence between time points (60’-40 and 40’-20’) was calculated (ΔE460/20minutes). The protein amount in the extract was calculated as described in 2.12.5 and the obtained values were used for the calculation of specific GUS activity [Ea(GUS)] according to this formula:

where:

m is the slope according to 4MU standard curve

The normalization of the GUS activity was done dividing the Ea(GUS) of a given sample by the Luciferase normalization factor, which was calculated by dividing the Luciferase activity of the sample by the average of the luciferase activity of all samples. With the normalized GUS values, the average and standard deviation were calculated for each construct and controls.