Appendix S1. Experimental Procedures
Cloning, expression and purification of recombinant ZmMYB31 DNA-Binding Domain
and ZmMYB31 unique region
PCR products containing the coding region for amino acids 10-116, 128-201 of
ZmMYB31 were first cloned into the pENTR/D entry vector and then recombined into
the pDEST17(N-term His), pDEST C1 (N-term His) and pDEST15 (N-term-GST)
expression vectors using Gateway® cloning technology (Invitrogen, CA). All plasmid
constructions were confirmed by DNA sequencing and expression constructs
transformed into the E. coli BL21-DE3 host. Cultures were grown to O.D.600 of 0.5-0.6,
induced for 3 h with 1 mM IPTG and harvested by centrifugation. Cells were
resuspended in 20 ml SB buffer and sonicated (6 times at 25% amplitude for 20 sec))
before affinity purification using Ni-NTA agarose resin or glutathione-sepharose resin
according to the manufacturer’s protocols (Qiagen, Germantown, MD). The eluted
proteins were then dialyzed and stored at -80°C.
AntiZmMYB31 Antiserum production and purification
The purified recombinant ZmMYB31 unique region protein was separated using
SDS-PAGE and stained with Coomassie blue. The band containing the protein was
excised (containing > 500 μg of proteins) and used for generating polyclonal antiserum
in rabbits (Cocalico Biologicals, Inc. Reamstown, PA). Two animals were immunized
with boost inoculations conducted at 14, 21 and 29 days post-immunization. To purify
the antiserum raised against ZmMYB31, the same region used for antiserum generation
was overexpressed in pDEST17 as a GST-fusion (GST-ZmMYB31 Unique). Polyclonal
antibodies specific for ZmMYB31 were purified following the method of Bar-Peled and
Raikhel (1996). The specificity of the purified antiserum was checked by western blot
using purified 6xHIS or GST tagged proteins and total proteins from E.coli and maize.
Approximately 50 ng of purified 6xHIS or GST tagged proteins and total proteins from
E.coli and maize plant were separated by electrophoresis on a 15% SDS polyacrylamide
gel and electro-transferred to PVDF membranes (BioRad). The membranes were probed
with non-purified serum (1:2,000 dilution) and affinity-purified antiserum (1:200
dilution).
Labelling DNA probes and electrophoretic mobility shift assays (EMSA)
The synthetic oligonucleotides used were ACII, ACIII, MBS, ABP1, APB5. ABP1
contains the high affinity P1 binding site (haPBS) from the maize A1 gene promoter,
and APB5, a mutated version in which the haPBS is destroyed (Grotewold et al., 1994).
End labelling of synthetic oligonucleotide probes was carried out using T4Polynucleotide Kinase (Invitrogen) in the presence of a two-molar excess of 32P-dATP
(>8,000Ci/mmol, GE Healthcare, Piscataway, NJ). The T4-Polynucleotide Kinase was
then heat killed by boiling for 20 minutes, followed by dilution of the labelled
oligonucleotides to a final volume of 50 µl in water. The labelled oligonucleotides were
then annealed to an equi-molar amount of complementary oligonucleotides by heating
to 95°C and cooling to room temperature. A fraction of the double-stranded labelled
oligonucleotides was precipitated on glass filters for quantification by scintillation of
the radiation incorporated. To precipitate the oligos, 2 µl of the oligo was pipetted onto
the glass filters and allowed to dry. The filters were then added to ~25 mL of 10% TCA
and 5 mM sodium phosphate buffer pH 7.5 and allowed to shake for 10 minutes at room
temperature. Next, the filters were washed with ~25 mL of 95% ethanol while shaking
for an additional 10 minutes at room temperature. Finally, a precipitated and nonprecipitated glass filter was quantified by scintillation.
For EMSA, DNA-binding reactions were performed essentially as described
(Grotewold et al., 1994) on ice for 30 minutes in a total of 25 µL with ~35 ng of
purified protein in A-0 buffer (10 mM Tris pH7.5; 50 mM NaCl; 1 mM EDTA; 5%
glycerol). The DNA-binding reactions also contained 0.8 µg of poly d(I)/d(C) and 1
mM dithiothreitol (DTT). After incubation on ice, 10,000 cpm of the oligonucleotide
DNA probes were added to each reaction and allowed to incubate on ice for an
additional 30 minutes. Protein-DNA complexes were resolved on a 8% polyacrylamide
gels (80:1 acrylamide:bis-acrylamide) in 0.25X TBE (22.5 mM Tris-borate and 0.5 mM
EDTA) running buffer at 415 V for 55 minutes at 4°C. The gels were then dried onto
Whatman® filter paper and subjected to autoradiography at –70°C overnight or a Kodak
phosphorimager for two hours and quantified using the BioRad imaging system
(Hercules, CA).
Chromatin immunoprecipitation (ChIP) experiments
ChIP experiments were performed as described previously (Morohasi et al., 2007).
Maize leaf sheath tissues (3 week old seedlings) were cross linked under vacuum for 15
min. Approximately 300 mg of tissue were ground for each immunoprecipitation. DNA
was sheared by sonication to approximately 300 bp fragments using Biorupter at
powder H for total 30 min. After preclearing with 40 µl of salmon sperm DNA/Protein
A-agarose beads (Upstate Biotechnology) for 2 hrs at 4°C, immunoprecipitation was
performed overnight at 4°C with either 2 µl of IgG, 5 µl of purified MYB31. After
reverse cross-linking of the immunoprecipitated and Input DNA (set aside from the
sonication step), the DNA was purified using the PCR Purification kit (Qiagen).
HPLC-UV-ESI-MS/MS determination of sinapoylmalate and flavonoids
Dry frozen leaves tissue from Arabidopsis thaliana (ca. 50 mg) were extracted with
5 mL of a mixture of MeOH/H2O (1:1 v/v) acidified with 0.8 % HCl 2N for 1h in an
Intelli mixer RM-2L (Elmi, Riga, Latvia) at room temperature. The mixtures were
centrifuged (3000g, 10 min), and the solid residue was extracted again with 2 mL
of acetone/water (3:7) for 1 h and centrifuged. The two supernatants were
combined, evaporated to dryness under a N2 stream, and diluted with 0.5 mL 0.1%
formic acid in water.
An Agilent series 1100 HPLC instrument (Agilent, Waldbronn, Germany) equipped
with a quaternary pump, an UV detector, and autosampler was used for HPLC-UV-ESIMS/MS experiments. Separation was conducted on a Phenomenex Luna C18(2)
(Torrance, CA, USA) 3.5 µm particle size column (50 x 2.1 mm i.d.) equipped with
Phenomenex Securityguard C18 column (4x 3 mm i.d.). Flow rate was 400µl/min, the
injection volume was 10 L and the separations were performed at 25 ºC. An API 3000
triple quadrupole mass spectrometer (PE Sciex, Concord, Ontario, Canada) equipped
with a TurboIon spray source was used to obtain MS/MS data. Settings were the
following: capillary voltage -3500 V (negative mode) or 5000 V (positive mode),
nebulizer gas (N2) 10 arbitrary units (a.u.), curtain gas (N2) 12 a.u., collision gas (N2) 10
a.u., declustering potential -30 V, focusing potential -200 V, entrance potential 10 V,
collision energy -30 V. The drying gas (N2) was heated to 400ºC and introduced at a
flow rate of 8000cm3 min-1. Full-scan data acquisition was performed, scanning from
m/z 100 to 1500 using a cycle time of 2 s with a step size of 0.1 units.
Different HPLC procedures were used for the analysis of the polyphenols. For
sinapoylmalate, a gradient elution was performed with a binary system consisting of:
[A] 0.1% aqueous formic acid and [B] 0,1% formic acid in acetonitrile. An increasing
linear gradient (v/v) of [B] was used, [t(min), %B]: 0,5; 30,23; 32,100; followed by a
re-equilibration step. For anthocyanin analysis the gradient elution was carried out with
in 5% formic acid in water [A] and 0.5% formic acid in acetonitrile [B] and the gradient
(v/v) was [t(min), %B]: 0,10; 20,40; 21,100; also with a posterior re-equilibration step.
Detection was carried out at 330 nm for sinapoylmalate, 265 nm for flavonols, and
520nm for anthocyanins. MS and MS/MS experiments were performed in negative
mode for sinapoylmalate and in positive mode for anthocyanins.
Protein extraction and 2D-PAGE
Seeds of transgenic and wild-type A. thaliana were germinated onto solid Murashige
and Skoog (MS) culture medium supplemented with 3% sucrose for one week. After
germination, seedlings were transferred into 250 ml flasks containing 50 ml liquid MS
medium supplemented with 3% sucrose (25 seedling/flask) and placed in a rotary
shaker (130 rpm) at 22ºC under a 16/8 h light/dark cycle for three weeks. At the end of
this culture period, roots from transgenic and control plants were collected, frozen in
liquid nitrogen and stored at -80ºC until analysed. 0.6 g of proteins were treated and
separated as previously described (Irar et al., 2006). 700 µg of total proteins were
loaded onto non-linear (NL) pH 3-11, 18 cm immobilized pH gradient (IPG) strips
(Immobiline DryStrips, Amersham Biosciences) for the first dimension. Bidimensional
electrophoresis was performed according to Campo et al., (2004) and gels were stained
with CBB G-250 (Bio-Rad). The experiment was repeated with two biological
replicates and three experimental replicates for biological sample. To evaluate protein
expression differences among gels, relative spot volume (% Vol.) was used. This is a
normalized value and represents the ratio of a given spot volume to the sum of all spot
volumes detected in the gel. Those spots showing a quantitative variation ≥ Ratio 1
were selected as differentially expressed. Statistically significant protein abundance
variation was validated by Student’s t-Test (p<0.05). The selected differential spots
were excised from the CBB G-250 stained gels and identified either by PMF using
MALDI-TOF MS or by peptide sequencing at the Proteomics Platform (Barcelona
Science Park, Barcelona, Spain). The MALDI-TOF MS analysis was performed
according to Oliveira et al., (2007).
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