Methods S1.
Experimental Procedures
Western blot analysis
Total protein was extracted from Arabidopsis leaves harvested from four week old plants by
grinding in 125 mM Tris–HCl (pH 8.8), 1% (w/v) SDS, 10% (v/v) glycerol, 50 mM Na2S2O5 and
centrifuging as described by (Martinez-Garcia et al., 1999). Protein concentration of the extracts
was determined using the BioRad protein assay (Bio-Rad, Hercules, California, USA), according
to the manufacturer’s protocol. 10 µg of total protein for each leaf extract and purified TyrA and
AtHPPD proteins were separated by SDS-PAGE on 12% gels, and transferred to Immobilon-P
membranes (EMD Millipore, Billerica, Massachusetts USA) in 25 mM Tris, 192 mM glycine,
10% methanol using a Mini-Genie blotter (Idea Scientific, Minnesota USA) at 12V for 30 min.
Membranes were probed with primary antibodies specific to AtHPPD or TyrA from rabbit
antisera (1:5000). Goat-anti-rabbit alkaline phosphatase was used as the secondary antibody
(Sigma, St. Louis, Missouri USA). The western blots were developed using BCIP/NBT alkaline
phosphatase substrate (Sigma, St. Louis, Missouri USA).
Polyclonal antibodies were prepared
from purified against purified E. coli expressed Arabidopsis HPPD and E. coli TyrA by Cocalico
Homogentisate measurement
The homogentisate content of plant tissues was measured according to the method described by
Karunanandaa et al. (2005) with modifications. The liquid chromatography/mass spectrometry
(LCMS) system consisted of a Shimadzu Prominence UPLC connected to a QTRAP4000
(ABSciex, Foster City, California USA) mass spectrometer. The system was operated using the
Analyst 1.5 software (ABSciex, Foster City, California USA). Chromatography was done using
an Eclipse Plus C18 column (2.1 x 50 mm, 1.8 micron particle size; Agilent Technologies,
Wilmington, Delaware USA) operated at a flow rate of 0.2 ml/min connected to the TurboV ion
source fitted with an electrospray ionization (ESI) probe. The mass spectrometer was operated in
negative ion mode, source temperature 300o C, and ESI needle voltage -4.5 kV (negative mode).
Optimized source/gas settings were determined by direct infusion of the homogentisate standard
(Sigma St. Louis, Missouri USA) at 30 ng/ul in methanol:water (50:50) + .1% formic acid at a
rate of .3 ml/hr. Optimized source/gas settings were: desolvation potential (DP) -60, Entrance
potential (EP) -5, Collision energy (CE) -17, Collision cell exit potential (CXP) -7, CAD gas
medium, Curtain gas (CUR) 20, gas1 85 arbitrary units, gas2 85 arbitrary units. The LC/MS/MS
method used mobile phases consisting of water +0.1% formic acid (A), and methanol + 0.1%
formic acid (B). The gradient started at 5% B (0 min) and increased to 98% B (1.6 min), held at
98% B until 1.8 min, and returned to 5% B at 2.8 min and was held at 5% until 5 min.
Homogentisate was monitored by the MRM transition 167.1/123.1 m/z. Under these conditions,
homogentisate eluted at 3.7 minutes. Injection volumes were 10 µl for all samples. Analytes
were quantified against external standard curves generated with the sample set. Homogentisate
solutions used to generate standard curves were dissolved in methanol:water (50:50) +.1%
formic acid. Standard curves were linear from 0.1 ng to 120 ng homogentisate. Quantitation of
homogentisate was done using Analyst 1.5 software.
Karunanandaa, B., Qi, Q., Hao, M., Baszis, S.R., Jensen, P.K., Wong, Y.-H.H., Jiang, J.,
Venkatramesh, M., Gruys, K.J., Moshiri, F., Post-Beittenmiller, D., Weiss, J.D. and
Valentin, H.E. (2005) Metabolically engineered oilseed crops with enhanced seed tocopherol.
Metabolic Engineering 7, 384-400.
Martinez-Garcia, J.F., Monte, E. and Quail, P.H. (1999) A simple, rapid and quantitative
method for preparing Arabidopsis protein extracts for immunoblot analysis. Plant J. 20, 251257.