Biochemical and physiological methods

2.3.1 Chlorophyll quantitation

Chlorophyll was extracted from inflorescences and leaves by incubating the detached uncrushed tissue in 96% (v/v) ethanol (10 mg of tissue per 30 μL of ethanol) for 4 d in the dark at 4°C. The chlorophyll content was then determined from 2 μL of the supernatant by measuring the absorbance at wavelengths 649 and 665 nm with a Nanodrop ND-1000 spectrophotometer and performing calculations as described by Wintermans and De Mots, (1965).

Chlorophyll a = 13.70 A665nm -5.76 A649nmμg mL-1

Chlorophyll b = 25.80 A649nm -5.76 A665nmμg mL-1

Total Chlorophyll = 6.1 A665nm +20.04 A649nmμg mL-1

The change in chlorophyll content from day 0 to the other days for each line or treatment was compared with that of wild-type Ler-0. One way ANOVA and Student’s t test were applied for statistical analysis of the regression parameter estimates.

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2.3.2 Protein quantitation

Reagents:

Extraction buffer: (100 mM Tris- HCl, pH 7.6, 10 mM MgSO4 and 10 mM dithiothreitol) Soluble proteins were extracted according to the method of Coupe et al. (2003). In brief, 50 mg of tissue was grounded in 500 μL of extraction buffer incubated on ice for 1 h with intermittent vortexing and the cellular debris was pelleted by centrifugation at 10,000 x g for 10 min at 4 °C. Soluble protein was then determined from the supernatant using the Coomassie dye-binding assay (Bio-Rad Laboratories) with bovine serum albumin as the standard.

2.3.3 Ion leakage

Relative electrolyte leakage was determined according to the method of Jing et al. (2002). Ten inflorescences (~90 mg of total weight) were harvested at each time point, placed in a 50 mL tube containing 10 ml of de-ionized water and incubated at 25°C for 30 min on an orbital shaker (200 rpm). The initial conductivity of the fluid was measured with Konduktometer model E527 (Metrohm Herisau). The total conductivity was determined after boiling the sample for 10 min. The relative electrolyte leakage was expressed as the percentage of the initial conductivity versus total conductivity.

2.3.4 Ethylene measurement

Ethylene was measured using a laser-based ETD-300 ethylene detector (Sensor Sense BV, Nijmegen, The Netherlands). Ethylene measurement was performed by using stop and flow method. In the stop and flow method selected cuvettes were flushed with air for 30 min to measure ethylene. Arabidopsis inflorescences (~100 mg) were removed and placed in a closed screw-capped 2 mL microfuge tube. The microfuge tube with inflorescences were connected to the instrument by first inserting two needles into the microfuge tube (to serve as inlet and outlet). Tubing was then fitted to the needles. The inlet tubing served to allow air from the catalyzer (that removes hydrocarbons) to enter the microfuge tube and the outlet

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tubing enabled the ethylene emanating from the tissue to move through a scrubber (to remove water and CO2) _{and then to the ETD for ethylene measurement. Ethylene production was}

measured in nL/h by using ethylene flow and accumulation.

2.3.5 JA and ACC measurement

JA and ACC concentrations were measured by using Liquid Chromatography- Mass Spectrometery (LC-MS) by Dr. Janine Cooney from Plant & Food Research Ruakura, Hamilton.

Preparation of Standards and Samples

A stock solution containing jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) each at 1000 μg mL-1 _was

prepared in methanol. A labelled internal standard stock solution containing the isotopically labelled analogues, [2_H

5]JA, [2H4]SA and [2H6]ABA each at 10 μg mL-1, was also prepared in

methanol. Calibration standards containing 0.1, 1, 10, 100 and 1000 ng mL-1 _{each of JA, SA,}

ABA and ACC were prepared by diluting the stock solution with 1:1 methanol:water. 25 ng of the labelled internal standard solution mix was added to 400 μL of each calibration standard. To 110 mg FW of freeze dried tissue on ice was added 25 ng of the labelled internal standard solution mix and 400 μL of the extraction solvent (20:79:1 methanol:isopropanol:acetic acid). The samples were shaken for 30 min at 4ºC and stored overnight at -20ºC. Following centrifugation at 4ºC and 13,000 x g for 10 min, a 100 μL aliquot of the supernatant was taken and diluted 10-fold with water before analysis by LCMS.

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LC-MS Method

LC-MS experiments were carried out on a 5500 QTrap triple quadrupole/linear ion trap (QqLIT) mass spectrometer equipped with a TurboIon-SprayTM interface (AB Sciex, Concord, ON, Canada) coupled to an Ultimate 3000 UHPLC (Dionex, Sunnyvale, CA, USA). Chromatographic separation was achieved on a Kinetex column (Phenomenex, Torrance, CA, USA) maintained at 60 ºC. Solvents were (A) water + 1% formic acid and (B)

acetonitrile + 0.1% formic acid and the flow rate was 600 μL min-1_{. For analysis of JA, SA}

and ABA, the initial mobile phase 15% B was ramped linearly to 50% B at 8 min, then ramped to 100% B at 8.1 min and held for 2 min before resetting to the original conditions.

Injection volume was 50 μL. MS data was acquired in the negative mode using a multiple reaction monitoring (MRM) method. For analysis of ACC the initial mobile phase 10% B was ramped linearly to 50% B at 10 min and held for 2 min before resetting to the original conditions. MS data was acquired in the positive mode using a multiple reaction monitoring (MRM) method. The transitions monitored, (Q1 and Q3), along with their optimised parameters (declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) are listed in Table 2.1.

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Table 2.1. MRM transitions used for plant hormones and their isotopically labelled internal standard analogues

Q1 Q3 Compounds DP EP CE C CXP 37.1 2.8 salicylic acid 140 10 22 - 9 09.0 9.0 jasmonic acid 25 10 25 - 15 63.0 53.0 abscisic acid 25 10 17 - 15 41.1 7.0 salicylic acid 35 10 30 - 15 14.0 2.0 jasmonic acid 25 10 25 - 15 69.0 59.0 abscisic acid 25 10 17 - 15 02.1 6.2 1-aminocyclopropane-1- carboxylic acid 0 0 5 1 5

Other operating parameters were as follows: dwell time, 60 ms; ionspray voltage, -4500 V for negative mode and 4500 V for positive mode; temperature, 600 ºC; curtain gas, 45 psi; ion source gas 1, 60 psi; ion source gas 2, 60 psi. Quantification was performed by the internal standard ratio method for JA, SA and ABA and by external calibration for ACC.

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