F. He et al. / Phytochemistry 117 (2015) 363–372
371
pore.com/) and analyzed by sodium dodecyl sulfate–polyacry-
supernatant was collected. The residues were re-extracted four
times. All of the supernatants were mixed, concentrated to dryness
using a rotary evaporator and then redissolved in EtOH: H2O
(10 ml, 1:9 v/v, pH 3.7). Each sample was independently extracted
twice.
lamide
gel
electrophoresis
(SDS–PAGE).
Total
protein
concentration was determined by the Bradford method using
bovine serum albumin (BSA) as the standard.
4.4. Recombinant Va5GT enzyme activity assays
4.7. HPLC–ESI–MS/MS analysis of anthocyanins
Typically, recombinant Va5GT protein (2.5
l
g), 1 mM donor
All of the resulting reaction mixtures and extracted suspensions
substrate (UDP-glucose or UDP-galactose) and 0.5 mM acceptor
substrate in K3PO4 buffer (100 mM, pH 7.0) were incubated at
25 °C for 20 min in a final volume of 100 ll. Different anthocyanins
were used as acceptor substrates, including pelargonidin-3-O-glu-
coside (2), cyanidin-3-O-glucoside (4), delphinidin-3-O-glucoside
(6), peonidin-3-O-glucoside (8), petunidin-3-O-glucoside (10),
malvidin-3-O-glucoside (12), cyanidin-3-O-rutinoside (14) and
malvidin-3-O-galactoside (16).
were filtered through 0.22 lm cellulose acetate and nitrocellulose
arately prior to HPLC–ESI–MS/MS analysis. Each sample was then
independently analyzed twice.
The analyses of anthocyanins in the enzyme assays and in the
grape skin extracts were carried out according to the method
reported by Han et al. (2008) with little alteration. An Agilent
with a G1379A Degasser, a G1311A QuatPump, a G1313A ALS, a
G1316A Column thermostat, a G1315B DAD and a Kromasil-C18
column (250 ꢀ 4.6 mm, 5 mm) was used. The solvents were as fol-
lows: (A) 2% HCO2H in H2O; and (B) MeCN containing 2% HCO2H.
The flow rate was 1.0 ml/min, and the solvent gradients used were
as follows: from 6% to 10% B over 4 min, from 10% to 25% B over
8 min, isocratic 25% B for 1 min, from 25% to 40% B over 7 min,
from 40% to 60% B over 15 min, from 60% to 100% B over 5 min
and from 100% to 6% B over 5 min. The injection volume was
For determination of optimum temperature for enzymatic
activity of Va5GT, 2.5
UDP-glucose and 0.5 mM malvidin-3-O-glucoside (12) in a final
assay volume of 100 l were incubated at 10 °C, 15 °C, 20 °C,
lg recombinant Va5GT enzyme, 1 mM
l
25 °C, 30 °C, 35 °C and 40 °C for 20 min. K2HPO4–KH2PO4 buffers
with a series of pH values, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and
11.0, were used to determine the optimum pH value for the enzy-
matic activity of Va5GT, and this assay was performed at 25 °C for
20 min. Then, the linear relationship between enzyme activity and
reaction time was assessed in a 100
ll reaction mixture consisting
30 ll, and the detection wavelength was 525 nm. The column tem-
of 500 M malvidin-3-O-glucoside (12) and 5 mM UDP-glucose in
l
perature was 50 °C. The MS conditions were as follows: ESI inter-
face, positive ion mode, 35 psi nebulizer pressure, 10 ml/min
drying N2 flow rate, 350 °C drying N2 temperature, capillary volt-
age 3000 V and scans at m/z 100–1000. All analyses were per-
formed in duplicate.
100 mM K3PO4 buffer (pH 7.0) incubated at 25 °C for different
times. For the determination of Va5GT kinetic parameters, all reac-
tion mixtures were incubated for either 6 min or 10 min at 25 °C
with a final reaction volume of 100
ll consisting of recombinant
Va5GT protein (2.5 g) and K3PO4 buffer (pH 7.0). For the acceptor
l
substrates, each assay contained 5 mM UDP-glucose with different
concentrations of malvidin-3-O-glucoside (12) ranging from 5 to
4.8. Analysis of the transcriptional expression of Va5GT during berry
development
600
with malvidin-3-O-glucoside (12) maintained at a constant con-
centration of 500 M and a UDP-glucose concentration that varied
from 0.05 to 2 mM.
lM. The UDP-glucose kinetic parameters were determined
RNA was extracted from the berry skins of ‘Zuoshanyi’ and
‘Zuohongyi’ grapes at all developmental stages. Then, RNA samples
were used for reverse transcription to produce the corresponding
cDNAs. The relative expression of Va5GT was monitored by real-
time PCR with the following gene specific primers: forward 50-
AGGTGATTGGAATTGGTTATGG-30 and reverse 50-GGCATTCTTTCTC
ATTTCTTGG-30. VvUbiquitin1 was chosen as the internal reference
(TIGR database: TC32075) and was amplified with the following
primers: forward 50-TGGTATTATTGAGCCATCCTT-30 and reverse
50-ACCTCCAATCCAGTCATCTAC-30. The amplification efficiency of
the primers used in the real-time PCR analysis was tested and
was found to be the same when serial dilutions were used. The
l
4.5. Subcellular localization of Va5GT
To observe the subcellular localization of Va5GT, the Va5GT cod-
ing sequence was amplified using the forward primer 50-
CCGCTCGAGATGGCGAATCCTCACCCCCA-30 and the reverse primer
50-ACGCGTCGACTGATAACCTTGTATAACCTCAT-30 (the restriction
sites for XhoI and SalI are underlined). The Va5GT ORF driven by
the Cauliflower mosaic virus (CaMV) 35S promoter was fused
upstream of GFP at the XhoI (50-end) and SalI (30-end) sites in the
pEZS-NL vector. A PYR1 protein tagged with mCherry was used
as the positive marker for the observation of Va5GT’s localization.
Protoplasts were isolated from 4-week-old Arabidopsis (ecotype
Columbia-0) leaves. The pEZS-NL-Va5GT and pMD19-T-PYR1
recombinant vectors were transiently transformed into protoplasts
by the PEG-Ca2+ method. The fluorescences of GFP and RFP were
observed separately using a confocal laser scanning microscope
(Nikon C1-Si, Japan) (Zhao et al., 2011).
PCR reaction tubes contained 5
Reference Dye, 4.5 l ddH2O, 1/6 l cDNAs and 1/3 l primers mix-
ture (10 M). The PCR parameters including pre-denaturation at
ll SYBR Green RT-PCR Master
l
l 50 ꢀ ROX
l
l
l
l
95 °C for 30 s and 40 cycles of 95 °C for 10 s and 60 °C for 30 s.
VvUbiquitin1 was chosen for the normalization of gene expression.
The gene expression measurements of Va5GT and VvUbiquitin1
were repeated in triplicate and were relatively quantified by the
D
2ꢁ
Ct
method (Bogs et al., 2005), with DCt equal to the cycle
threshold of Va5GT – the cycle threshold of VvUbiquitin.
4.6. Anthocyanin extraction from grape skins
Acknowledgements
According to a previously published method, the skin was
peeled and immediately ground into a powder in liquid N2 (He
et al., 2010b). Grape skin powder (0.50 g) was immersed in
MeOH (10 ml) containing 2% HCO2H. The mixture was ultrasound
sonicated for 10 min followed by shaking in the dark at 25 °C for
30 min at 150 rpm. The homogenate was centrifuged and the
We greatly thank Prof. Wilfried Schwab (Biomolecular Food
Technology, Technische Universität München, Germany) for pro-
viding us with the cDNA sequences of Cha5GT and Dia5GT. We
thank Prof. Da-Peng Zhang (Protein Science Laboratory of the
Ministry of Education, School of Life Sciences, Tsinghua
He, Fei
