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be inhibitory when applied at high concentrations. Further, the growth-promotive
or inhibitory effects of exogenous SA application appear to depend on the envi-
ronmental conditions at the time of application (Hayat et al.
2010
). Based on the
above literature, we think it is time to evaluate the effects of environmental sig-
naling on endogenous SA levels in the context of a range of plant growth
mechanisms. Such an approach should allow us to better understand how exoge-
nously applied SA alleviates stress symptoms in many higher plants.
2 Assessing Endogenous Salicylic Acid Levels
Endogenous SA levels in plant tissues can be precisely identified and accurately
quantified using the stable isotope dilution method (Gaskin and MacMillan
1991
).
This is accomplished by selected ion monitoring (SIM) using capillary gas chro-
matography (GC)—mass spectrometry (MS) or liquid chromatography (LC)—MS
(Scott et al.
2004
; Kurepin et al.
2010a
). The first stage, consisting of extraction,
addition of stable isotope internal standards and purification is the same for both
SIM-GC-MS and LC-MS methods. Plant tissue is collected and immediately
frozen in liquid N
2
, then freeze-dried or stored at -80 8C until extraction.
Extraction consists of grinding in a mortar and pestle with liquid N
2
, followed by
use of 80 % aqueous methanol (H
2
O-MeOH = 20:80, v/v) as an extraction sol-
vent. A known amount of high specific activity deuterated SA (usually
2
H
6
-SA) is
added to the 80 % MeOH extract as a quantitative internal standard. The 80 %
MeOH extract is then purified with a C
18
preparative column (using reversed phase
C
18
material) and dried in vacuo at 35 8C. For the LC-MS method, the dried
residue is reconstituted in 0.05 % HOAc in H
2
O-MeCN (85:15, v/v) and then
filtered with a 0.45 lm filter prior to the injection into the LC-MS. The charac-
teristic m/z ions that are usually used for quantification are (m/z 141 for d
6
SA and
m/z 137 for the endogenous protio SA). The quantification of endogenous SA is
then accomplished based on the relative intensities (peak areas) of these charac-
teristic m/z ions, the known amount of d
6
-labeled SA internal standard added and
the recorded fresh or dry tissue weight (Scott et al.
2004
). For the SIM-GC-MS
method, the dried residue is reconstituted in 1 % HOAc in H
2
O-MeOH (90:10, v/
v) and injected into the high performance LC (HPLC). In one example (Kurepin
et al.
2010a
) the HPLC used a manually implemented 10-73 % linear gradient
program for separation of SA from other plant metabolites. The eluted fractions
that are expected to contain SA (based on previous HPLC runs with a SA standard)
are dried in vacuo at 35 8C and then methylated using ethereal CH
2
N
2
at room
temperature as described in Kurepin et al. (
2010a
). The methylated SA sample is
then injected into the GC-MS and the SIM program is set to monitor characteristic
m/z ions for both the deuterated SA-Me standard (124, 96 and 156 for d
6
SA) and
endogenous protio SA-Me (120, 92 and 152). For identification of endogenous SA
a comparison of the relative intensities of the three m/z ion pairs, i.e. 124/120, 96/
92 and 156/152, is accomplished. The amount of endogenous (protio) SA is then
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