Biochemical analysis 47

The leaf phosphate content and the root acid phosphatase activity were determined from the individual plant material from the final destructive harvest of each experiment.

2.4.1. Quantification of leaf phosphate

A standard curve containing 0 to 200 µM KH2PO4 (MW 136.1 g/mol) was used to

The frozen pre-weighted leaves were ground in three volumes of 5 M H2SO4 in a 1.5 mL

eppendorf®

tube with an eppendorf®

grinder. The mixture was vortexed, and then centrifuged at 13,000g for 10 minutes (min). The supernatant was diluted by removing 100 µL of liquid and added to 1 mL of MilliQ water. A stock solution of concentrated phosphate reagent containing 16 mM (NH4)6(Mo7O2)4, 0.15 mM antimony potassium

tartrate oxide, and 2.25 mM H2SO4 was made to use for the phosphate reaction mix.

Immediately prior to testing, the phosphate reaction mix for 50 reactions was made and contained 1.25 mL concentrated phosphate reagent, 5 mL of 197.58 mM ascorbic acid and 3.75 mL MilliQ water in a total of 10 mL. To test the leaf P content, duplicate assays of each leaf contained 10 µL of the diluted sample, 40 µL of MilliQ water, and 200 µL of phosphate reaction mix in a total volume of 250 µL.

OD

620 nm

y = 0.0052x- 0.0156

R² = 0.99874

Concentration KH2PO4 µM

Figure 2.3. Standard curve for 200 mM KH2PO4

The concentration of phosphate in the leaves was determined by a standard curve of KH2PO4 ranging from to 200 µM in a total volume of 250 µL.

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2.4.2. Acid phosphatase activity of root soluble and cell wall fractions Extraction procedure

All of the frozen root material was ground in liquid nitrogen and a sample of approximately 300 µg was transferred and weighed into an eppendorf®

tube. The root samples were resuspended in three volumes of 1 mM dithiothreitol (DTT), and then centrifuged at 6,000g for 10min. The supernatant was removed and designated the soluble fraction, and stored at 4℃ overnight for analysis of acid phosphatase activity the following day. The pellet was washed with 100 µL of 1 mM DTT and centrifuged at 6,000g for five min, the supernatant discarded and the pellet washes were repeated another two times, with the last wash centrifuged at 13,000g for five min. The pellet was then washed with 100 µL of MilliQ water three times and centrifuged at 13,000g for five min each time, before being extracted overnight at 4℃ with 1 M NaCl.

The amount of NaCl was approximately 50 µL and was just enough to cover the surface of the pellet. The next day, the suspended pellet was centrifuged at 13,000g for five min and the supernatant was collected and designated the cell wall fraction. The pellet was then resuspended in 1 M NaCl and incubated at 37℃ for one hour. The suspended pellet was then centrifuged at 13,000g for five min and the supernatant was collected and combined with the cell wall fraction that was previously collected.

Enzyme activity

The acid phosphatase assay was modified from the method used by Zhang and McManus (2000). The 5.4 mM p-nitrophenyl phosphate (pNPP) substrate buffer was freshly made by dissolving one 5 mg tablet of pNPP in 2.5 mL of 0.1 M sodium citrate buffer (pH 5.6). The acid phosphatase activity was measured in duplicate by adding 20 µL of the soluble and cell wall fractions to 80 µL of MilliQ water and 100 µL of substrate buffer. The samples were incubated at 37℃, the absorbance read at 405 nm every min for a total of 30 min. The activity of acid phosphatase was calculated from the amount of p-nitrophenol (pNP) formed in 30 min from a standard curve ranging from 0 µM to 500 µM pNP (Figure 2.4). The soluble protein was determined (Figure

△ OD 405 nm /m inu te y = 0.0038x - 0.0039 R² = 0.99817 Concentration of p-nitrophenol (µM)

Figure 2.4. Standard curve for p-nitrophenol

Standard curve for p-nitrophenol. The enzyme assay was measured over 30 min at 405 nm and the change was calculated to give a rate per minute.

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2.4.3. Quantification of soluble protein

A standard curve was used to quantify the amount of soluble protein in 1 mg to 5 mg of bovine serum albumin (BSA) (Figure 2.5). The microtitre plates contained the various amounts of BSA in 160 µL with 40 µL of Bio-Rad protein dye then added to give a final volume of 200 µL and the absorbance read at 595 nm in a 96-well Bio Strategy microtitre plate reader at room temperature. The quantity of soluble protein in the soluble and cell wall fractions of the roots used to determine the acid phosphatase activity described in section 2.4.2 was then determined. The assay contained 10 µL of protein extract with 150 µL Milli-Q water and then 40 µL of Bio-Rad protein dye.

OD

595 nm

y = 0.1232x + 0.0281 R² = 0.98924

Bovine serum albumin mg/mL

Figure 2.5. Standard curve for 1 mg/mL to 5 mg/mL bovine serum albumin

A standard curve of Bovine Serum Albumin (BSA) ranging from 1 mg/mL-5 mg/mL to quantify the amount of soluble protein in 200 µL assay containing 40 µL and 160 µL protein.

The absorbance was read at 595 nm in a 96-well Bio Strategy microtitre plate reader at room temperature and the curve was constructed.

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