H.K. Seneviratne et al. / Phytochemistry xxx (2014) xxx–xxx
7
of 0.2 ml minꢁ1; linear gradient of A: B from 90:10 to 60:40 in
6.5 min, to 20:80 in 2 min, to 0:100 in 1 min followed by 1 min
at 0:100, and finally to 90:10 in 0.5 min with this being held for
4 min. The column temperature was kept at 25 °C and sample
to a neutral Al2O3 column and eluted with EtOAc–MeOH–H2O,
350 ml, 200: 33: 27 (v/v). After some unreacted aglycone (3)
(28 mg, 31.5%) was eluted first, pinoresinol-40-O-b-
D-monogluco-
side (4) was collected, with the corresponding eluate evaporated
to give a clear oil which slowly solidified as an amorphous product
(42.5 mg, 33%). MS, 1H, 13C NMR, and UV spectroscopic analyses of
injection volume was 5 ll. Masses were determined using a Waters
Xevo G2 qToF mass spectrometer, using leucine-enkephalin as a
lock-mass standard, at a capillary voltage of 3 kV, cone voltage of
38 V, a desolvation gas temperature of 280 °C and source temper-
ature of 100 °C. Masslynx V4.1 (Waters Corp.) was used to collect
and process data. The chemical identities of the compounds
observed were confirmed by comparing their MS/MS and MSn
spectra (fragmentation pattern) with authentic standards. Identifi-
cation of pisatin (1), pinoresinol (3) and pinoresinol monoglucoside
(4) was done by comparing the retention time, UV, and MS spectra
of authentic standards.
UPLC–ESI-qToF–MS coupled with ion mobility mass spectrome-
try experiments were performed on a Synapt G2 high-definition
mass spectrometer (Waters Corp., Manchester, UK), a hybrid quad-
rupole/ion mobility/orthogonal time-of-flight mass spectrometer.
The traveling wave ion mobility device employs dynamic electric
fields under reduced pressures, with a trap and transfer cell located
in front of, and after, the ion mobility separator, respectively. Col-
lision-induced dissociation (CID) was initiated in the transfer cell
by elevating collision energy (CE). A wave height of 40 V and a
wave velocity of 1000.0 msꢁ1 were utilized for ion mobility separa-
tions in both positive and negative ion modes. He was used as a
drift gas at a flow rate of 90 ml minꢁ1, resulting in a pressure of
3.5 mbar in the ion mobility device. Masslynx V4.1 (Waters Corp.)
was used to collect and process data. Chemical identity of metab-
olites was confirmed by comparing ion mobility MSn spectra (frag-
mentation pattern) with authentic standards.
pinoresinol-40-O-b-
D-monoglucoside (4) were in agreement with
published data (Vermes et al., 1991). The coupling constant
(J = 7.5 Hz) of the anomeric H atom confirmed that the glucopyran-
osyl unit existed in a b-configuration.
4.9. Gene expression analysis
Total RNA (5
fungally infected pea pods using the RNeasy plant Minikit (Qiagen),
with first strand cDNA (1 g) synthesized using the SuperScript™
lg) was individually isolated from uninfected and
l
III First-Strand Synthesis System for RT-PCR (Invitrogen). For real
time quantitative PCR analyses, each PCR mixture contained syn-
thesized first strand cDNA, Platinum SYBR Green qPCR SuperMix-
UDG (Invitrogen) and gene-specific primers, which were designed
using Primer Premier software (Biosoft International, Palo Alto, CA)
(Supplemental Table S1). Expression levels were normalized
against the pea actin gene with expression levels for the DRR206
gene in uninfected pea pods set to 1 and data averaged from trip-
licate samples.
4.10. Cloning and construction of the DRR206 expression vector
Vector construction and transformation were carried out as
described by Kim et al. (2012). The DRR206 gene (GenBank
U11716) was PCR amplified using gene specific primers
(Forward 50-ATGGGTTCCAAACTTCTAGTACTA-30 and Reverse 50-
TTACCAACACTCAAAGAACTTGAT-30) from genomic DNA isolated
from pea leaves. The amplified fragment was then subcloned into
the pCR4TM-TOPOÒ vector. The DRR206 sequence was re-amplified
with the linker primers (Forward 50-GGAATTCATGGGTTC-
CAAACTTCTA-30 and Reverse 50-GAAGCTTTTACCAACACTCAA-
AGAA-30) which harbored restriction enzyme sites designed for
directional cloning into the pART7 vector. After digestion of PCR
products with EcoRI and HindIII, the DNA fragments were ligated
with pART17 vector which was also digested with the same restric-
tion enzymes. After sequence verification, the biolistic bombard-
ment technology was employed to introduce the DRR206-
harboring pART17 into tomato (S. peruvianum) cells (Kim et al.,
2012). Tomato calli were produced on media containing kanamy-
4.6. Isolation of pinoresinol (3) from fungally infected pea pods
The EtOAc extract from fungally infected pea pods was sequen-
tially purified by preparative silica gel TLC (eluant, CH2Cl2–MeOH,
9:1, v/v) and then by reversed-phase HPLC to afford pinoresinol (3),
with the latter subsequently subjected to chiral HPLC analysis (see
‘‘Section 4.1’’).
4.7. Isolation of pinoresinol-40-O-b-
-monoglucoside (4) from fungally
D
infected pea pods
Fungally infected pea pods (2.4 g), as above, were subjected to
metabolite extraction using MeOH–H2O (70:30, v/v) (3 ꢂ 400 ml),
with extracts centrifuged (3000ꢂg for 5 min) and evaporated to
dryness in vacuo. Further purification was done using a reversed-
phase HPLC system. Purified fractions were combined and sub-
jected to reversed-phase UPLC–MS and ion mobility mass spectro-
metric analyses, as described above (see ‘‘Section 4.5’’).
cin (75 l
g ml-1) and the gene expression level of DRR206 in each
tomato callus was assessed using RT-PCR. Six calli preparations
were selected for cell suspension culture production of the
DRR206 recombinant protein.
4.11. Heterologous expression and purification of DRR206 in plant cell
cultures
4.8. Chemical synthesis of pinoresinol-40-O-b-
-monoglucoside (4)
D
This was carried out as previously described (Vermes et al.,
1991) with the following modifications. To a solution of ( )-pino-
resinols (3) (89 mg, 0.25 mmol) in Me2CO (6 ml) and 2.5% KOH
Expression and purification of the DRR206 recombinant protein
in tomato cell culture were carried out according to the procedures
described in Kim et al. (2012). Transformed suspension cell cul-
tures (40 ml each) were gradually scaled-up weekly by inoculating
them into new media (up to 3 l). Seven days after final inoculation,
cells were harvested by vacuum filtration. ‘‘Cell wall bound’’ pro-
teins were recovered after stirring the cells in KPi buffer (0.1 M,
pH 5.9; buffer A) first containing 75 mM KCl and then 150 mM
KCl. After agitating the cell suspension for 30 min at 100 rpm at
4 °C, both buffered solutions were recovered by vacuum filtration,
combined and mixed with SP-Sepharose fast flowÒ resin (80 ml)
pre-equilibrated with buffer A containing 75 mM KCl. Proteins
were eluted with buffer A containing 1 M NaCl, followed by (NH4)2-
(0.68 ml, 0.3 mmol), a solution of 2,3,4,6-tetra-O-acetyl-a-D-gluco-
pyranosyl bromide (127.5 mg, 0.3 mmol) in Me2CO (4 ml) was
added dropwise at 0 °C. After stirring for 1.5 h at 0 °C and standing
overnight at 5 °C, the solvent was then evaporated in vacuo, with
the resulting residue diluted with H2O (5 ml) and the whole
extracted with EtOAc (10 ml). The resulting residue so-obtained
was dissolved in MeOH (20 ml), with the solution adjusted to pH
10 with 1 M NaOMe and left standing overnight at room tempera-
ture. After neutralization with Amberlite IR-120 cation exchange
resin, filtration and evaporation, the resulting residue was applied
Please cite this article in press as: Seneviratne, H.K., et al. Non-host disease resistance response in pea (Pisum sativum) pods: Biochemical function of
Seneviratne, Herana Kamal
