M. P. López-Gresa et al.
The inoculation procedure (Bellés et al., 1991) of Rutgers tomato plants
8 days after sowing (cotyledon stage) with citrus exocortis viroid (CEVd)
was performed by puncturing the stems of the seedlings with a needle
dipped in either buffer or the 2 M LiCl-soluble fraction of nucleic acids
from CEVd-infected tissue, as described previously (Semancik et al., 1975).
The control and infected tomato plants were placed in a controlled
growth room at 30/25°C (16 h day/8 h night) with 70% relative humidity.
Symptoms appeared 15–17 days after viroid infection. Control and
infected tomato leaves were recollected 2 and 4 weeks after infection,
frozen under liquid nitrogen, homogenised using precooled mortar and
freeze-dried. Three biological replicates were analysed for each time.
defence. Different metabolic profiles are indicative of changes in
metabolic pathways, thus, for example, enabling health and
disease conditions in a system to be distinguished (Hankemeier,
2007). Comparing metabolic profiles of infected plants vs the
corresponding controls is a powerful tool to unravel the bio-
chemical pathways involved in multi-factorial disorders. Plants
produce a huge number of metabolites which may largely differ
in chemical characteristics. For this reason, a combination of
methods to prepare and analyse samples is required. Thus, an
extraction system and an instrumental technique must be
carefully chosen to suit a particular biological question. Typical
analytical equipment employed in metabolomics includes
chromatographic methods [liquid chromatography (LC), gas
chromatography (GC), capillary electrophoresis, and thin layer
chromatography], mass spectrometry (MS) and nuclear magnetic
resonance (NMR) spectroscopy (Verpoorte et al., 2007).
In this study, a direct extraction method followed by NMR spec-
troscopy analysis in combination with multivariate data analysis
was employed in order to investigate the metabolites associated
with two different infections in tomato plants. Identification of
metabolites responsible for the differences between control and
infected samples can provide information about the chemical
diversity of the signalling compounds involved in the defence
response in plant–pathogen interaction.
Extraction and NMR spectra measurements
Fifty milligrams of freeze-dried plant material were extracted in 2 mL
Eppendorf tubes with 1.5 mL of a mixture of KH2PO4 buffer (pH 6) in D2O
containing 0.05% trimethylsilyl propionic acid sodium salt (TMSP) and
CH3OH-d4 (1:1). The extracts were vortexed vigorously, sonicated for
20 min and then centrifuged at 13000 rpm for 10 min. Eight hundred
microlitres of the supernatant was transferred in 5 mm NMR tubes for the
spectral analysis.
1H-NMR, 2D-J resolved, 1H–1H correlated spectroscopy (COSY), and het-
eronuclear multiple bonds coherence (HMBC) spectra were recorded at
25ºC on a 600 MHz Bruker AV 600 spectrometer equipped with cryo-
probe operating at a proton NMR frequency of 600.13 MHz. Methyl
signals of CH3OH-d4 were used as the internal lock. Each 1H NMR spectrum
consisted of 128 scans requiring 10 min acquisition time with the follow-
ing parameters: 0.25 Hz/point, pulse width (PW) = 30° (10.8 μs), and
relaxation delay (RD) = 1.5 s. A presaturation sequence was used to sup-
press the residual H2O signal with low power selective irradiation at the
H2O frequency during the recycle delay. FIDs were Fourier transformed
with LB = 0.3 Hz and the spectra were zero-filled to 32 K points. The
resulting spectra were manually phased and baseline-corrected, and cali-
brated to TMSP at 0.0 ppm, using Topspin (version 2.1, Bruker). All the 2D
NMR parameters were the same as in our previous reports (Jahangir
et al., 2008).
Experimental
Plant material and inoculation procedure
Seeds from tomato (Solanum lycopersicum cv. Rutgers) (Western Hybrid
Seeds Inc., CA, USA) were used in the experiments. The plants (one per
pot) were grown in 15 cm-diameter pots containing a mixture of peat
(Biolan) and vermiculite 1:1. The pots were subirrigated with a nutrient
solution as described by Naranjo et al. (2003).
Infection of 5-week-old tomato plants with P. syringae pv. tomato was
performed with a bacterial suspension obtained as follows: bacteria were
grown overnight at 28°C in 20 mL Petri dishes with C3 agar medium
(Oxoid, Basington, UK) supplemented with 0.45 g of KH2PO4 per litre and
2.39 g of Na2HPO4.12H2O (pH 6.8) per litre. Bacterial colonies were then
resuspended in 10 mM MgSO4 to a final concentration of OD600: 0.1.
Dilution plating was used to determine the final inoculum concentration,
which averaged 107 cfu/mL. Two types of bacterial infections were carried
out by infiltration and immersion. The bacterial infiltration procedure was
as described in detail by Collinge et al. (1987). Briefly, aliquots of 100 μL
of this bacterial suspension were injected into the abaxial side of each
leaflet (three to four panels per leaflet averaging 30 mm2) of the third and
fourth leaf from the base of the plant with a 1 mL sterilised plastic syringe
without needle. Equivalent control leaflets were mock-inoculated with
10 mM MgSO4. The hypersensitive reaction consisted in the appearance
of necrotic brown spots and cellular death in the inoculated leaf surface
area 24 h after bacterial infiltration. Also, a strong epinasty of the inocu-
lated leaves was evident at this time in response to the biotic stress
(Zacarés et al., 2007). For the bacterial immersion infection, the aerial
portions of tomato plants were dipped in the suspension of bacteria
(107 cfu/mL) containing 10 mM MgSO4 and Silwet L-77 (0.05%) for 20 s,
as described previously (Martin et al., 1993). No symptoms were observed
in these bacterial-treated plants when the samples were collected. For
mock inoculations, plants were dipped in buffer with Silwet L-77 (0.025%)
alone. The tomato plants were maintained in the greenhouse at 27 and
23°C (16 h day and 8 h night, respectively) and with relative humidity
from 50 to 70%. Then the third and fourth leaves were harvested 48 h
after bacterial infection by immersion or infiltration. The leaves were
immediately frozen under liquid nitrogen, subsequently ground in a
mortar and lyophilised. Three biological replicates were analysed for each
treatment.
Data analysis
1H NMR spectra were automatically reduced to ASCII files using AMIX
(version 3.7, Bruker Biospin). Spectral intensities were scaled to total
intensity TMSP and reduced to integrated regions of equal width
(0.04 ppm) corresponding to the region of δ 0.4–10.00. The region of δ
4.7–4.9 was excluded from the analysis because of the residual signal of
water as well as δ 3.28–3.34 for residual methanol. Principal component
analysis (PCA) and partial least square-discriminant analysis (PLS-DA)
were performed with the SIMCA-P software (version 11.0, Umetrics,
Umeå, Sweden) using Pareto or unit variance (UV) scaling method.
Extraction and LC-MS analysis
Leaflets (0.5 g fresh weight) of tissue were ground with a pestle in a
mortar using liquid nitrogen, then homogenised in 1.5 mL of 90% metha-
nol. The extracts were vortexed vigorously, sonicated for 15 min and then
centrifuged at 14000 g for 15 min using 2 mL Eppendorf tubes to remove
cellular debris. The supernatant (1.5 mL) was dried at 35°C under a flow
of nitrogen. The residue was resuspended in 100 μL of methanol and
filtered through 0.45 μm Spartan 13/0.45RC filters (Schleicher & Schuell,
Keene, NH, USA) nylon filters (Waters, Millford, MA, USA). The samples
were analysed in electrospray ionisation (ESI)-MS using a 1515 Waters
HPLC binary pump, a 996 Waters photodiode detector (range of maxplot
between 240 and 400 nm, spectral resolution of 1.2 nm), and a ZMD
Waters single quadrupole mass spectrometer ESI ion source. The source
parameters of the mass spectrometer for ESI in positive mode were the
following: capillary voltage 2500 V, cone voltage 20 V, extractor 5 V, RF
Lens 0.5 V, source block temperature 100°C and desolvation gas tempera-
ture 300°C. The desolvation and cone gas used was nitrogen at a flow of
Copyright © 2009 John Wiley & Sons, Ltd.
Phytochem. Anal. 2010, 21, 89–94