Group Title: Plant Methods 2008, 4:28
Title: A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid
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Title: A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid
Series Title: Plant Methods 2008, 4:28
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Creator: DeFraia CT
Schmelz EA
Mou Z
Publication Date: 39813
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Plant Methods
Plant Methods goMed Central


Methodology I


A rapid biosensor-based method for quantification of free and
glucose-conjugated salicylic acid
Christopher T DeFraia', Eric A Schmelz2 and Zhonglin Mou*1


Address: 'Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL, 32611, USA and 2Center for
Medical, Agricultural and Veterinary Entomology, United States Department of Agriculture, Agricultural Research Service, 1700 SW 23rd Drive,
Gainesville, FL 32608, USA
Email: Christopher T DeFraia cdefraia@ufl.edu; Eric A Schmelz eric.schmelz@ars.usda.gov; Zhonglin Mou* zhlmou@ufl.edu
* Corresponding author



Published: 31 December 2008 Received: 21 July 2008
Plant Methods 2008, 4:28 doi: 0.1 186/1746-4811-4-28 Accepted: 31 December 2008
This article is available from: http://www.plantmethods.com/content/4/1/28
2008 DeFraia et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Salicylic acid (SA) is an important signalling molecule in plant defenses against
biotrophic pathogens. It is also involved in several other processes such as heat production,
flowering, and germination. SA exists in the plant as free SA and as an inert glucose conjugate
salicylicc acid 2-O-p-D-glucoside or SAG). Recently, Huang et al. developed a bacterial biosensor
that responds to free SA but not SAG, designated as Acinetobacter sp. ADPWH_Iux. In this paper
we describe an improved methodology for Acinetobacter sp. ADPWH_lux-based free SA
quantification, enabling high-throughput analysis, and present an approach for the quantification of
SAG from crude plant extracts.
Results: On the basis of the original biosensor-based method, we optimized extraction and
quantification. SAG content was determined by treating crude extracts with P-glucosidase, then
measuring the released free SA with the biosensor. P-glucosidase treatment released more SA in
acetate buffer extract than in Luria-Bertani (LB) extract, while enzymatic hydrolysis in either
solution released more free SA than acid hydrolysis. The biosensor-based method detected higher
amounts of SA in pathogen-infected plants than did a GC/MS-based method. SA quantification of
control and pathogen-treated wild-type and sid2 (SA induction-deficient) plants demonstrated the
efficacy of the method described. Using the methods detailed here, we were able to detect as little
as 0.28 tig SA/g FW. Samples typically had a standard deviation of up to 25% of the mean.
Conclusion: The ability of Acinetobacter sp. ADPWH_Iux to detect SA in a complex mixture,
combined with the enzymatic hydrolysis of SAG in crude extract, allowed the development of a
simple, rapid, and inexpensive method to simultaneously measure free and glucose-conjugated SA.
This approach is amenable to a high-throughput format, which would further reduce the cost and
time required for biosensor-based SA quantification. Possible applications of this approach include
characterization of enzymes involved in SA metabolism, analysis of temporal changes in SA levels,
and isolation of mutants with aberrant SA accumulation.







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Background
The plant signal molecule salicylic acid (SA) has been
shown to play a role in several physiological processes,
including heat production, flowering, germination and
pathogen resistance [1-5]. In the last two decades, its role
in pathogen resistance has been studied extensively [6,7].
Treatment with SA confers resistance to a variety of bio-
trophic pathogens [5,8], and pathogen infection causes
the accumulation of SA [9,10]. SA can be glucosylated to
form SAG (2-0-p-D-glucosylsalicylic acid), which serves
as a biologically inert reservoir of SA [11]. SA is also
present in plants as methyl-salicylate, which can also be
conjugated to glucose [12]. Generally, mutants with con-
stitutively high SA levels are resistant to biotrophic patho-
gens, while those unable to accumulate SA are susceptible
[13-24]. Thus, quantification of SA is routine in the study
of plant immunity.

The most commonly used methods for measuring SA
from plant tissue employ HPLC or GC/MS [25-27]. These
techniques both involve extraction of SA in organic sol-
vents and subsequent evaporation. SA is then purified
chromatographically, and detected by fluorescence spec-
troscopy or mass spectrometry. However, during extrac-
tion some of the SA is lost, and an internal control must
be included to correct for SA recovery.

Recently, Huang et al. developed a biosensor for SA,
named Acinetobacter sp. ADPWH_lux [28]. This strain is
derived from Acinetobacter sp. ADP1, and contains a chro-
mosomal integration of a salicylate-inducible luxCDABE
operon, providing the substrate and catalyst for SA-
responsive luminescence. The Acinetobacter sp.
ADPWH_lux response appears to be limited to SA,
methyl-SA, and the synthetic SA derivative acetylsalicylic
acid [28]. Measurement of SA from TMV-infected tobacco
leaves with the biosensor and GC/MS yielded similar
results [29], demonstrating that this strain is suitable for
the quantification of SA from plant tissue.

Herein, we present an improved approach for the quanti-
fication of free SA from Arabidopsis leaf extracts using Aci-
netobacter sp. ADPWHlux. We have also developed a
method for Acinetobacter sp. ADPWH_lux-based SAG
measurement.

Results
Standard Curve Generation
Briefly, the method described by Huang et al. comprises
tissue grinding, extraction in LB, sonication, and centrifu-
gation, resulting in a crude plant extract containing SA.
The crude extract is then mixed with a culture of the bio-
sensor in a 96-well cell culture plate, and incubated at
37C for one hour. The luminescence is then determined.
In order to convert SA-induced luminescence into units of


SA concentration, several standards with known amounts
of SA are included to generate a standard curve [28]. We
found that standards made with crude extract had signifi-
cantly lower luminescence than those made with LB (Fig-
ure 1A), suggesting that the plant extract decreases
induction of the biosensor by SA. Since our aim was to
determine SA concentrations in plant extract, the stand-
ards must also have plant extract as a solvent. The ideal
plant extract for making SA standards would initially con-
tain no SA. In order to minimize the SA content of the
extract used to make the standards, we used extract from
sid2-2 plants, which fail to accumulate significant
amounts of SA during pathogen infection. However, we
and others [24] were unable to consistently detect a differ-
ence in constitutive SA levels between sid2-2 and wild type
(data not shown). Therefore, untreated wild type plants
may also be used for making the SA standards. Lack of a
standard with no SA precludes the determination of abso-
lute SA concentrations from plant extracts. Thus, the bio-
sensor may only be used to determine relative SA levels
between samples rather than absolute concentrations.
When SA standards were made with plant extract, the rela-
tionship between luminescence and SA concentration was
non-linear (Figure 1B). To simplify data analysis, instead
of using all standards to construct the standard curve, only
the standards with luminescence similar to that of the
experimental sample were used. A best-fit linear line with
a high R-squared value could then be derived and used as
the standard curve (Figure 1C). Alternatively, a non-linear
best-fit line can be used, although we found higher R-
squared values for standards with low SA content, using
the former method. Conversion from luminescence to SA
concentration was carried out using the following equa-
tion:

[SA] = [(luminesence y-interceptstandard curve)/slopestandard
cunvl/tissue mass

where known luminescence of a sample and tissue mass
are used to calculate unknown SA concentration. In some
cases, two or more standard curves were needed to deter-
mine the SA concentration of samples with largely differ-
ent luminescence values. We found this approach to be
useful in determining SA content between 1.6 and 64 ng
SA (0.28 and 11 itg SA/g FW). At higher concentrations,
induction of the biosensor by SA was diminished (Figure
ID). If sample SA concentrations exceeded 11 itg SA/g FW,
the sample extract was diluted in untreated plant extract
so that it fell within the useful range of the assay.

To determine if the culture density of the biosensor
affected the useful range of the assay, we tested cultures of
various optical densities (ODs) for SA-induced lumines-
cence. The responsiveness ofAcinetobacter sp. ADPWH_lux
increased with culture density, reaching a maximum at


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SLB
0 plant extract I




44
Se 9
Q 9 '

S10 20 30 40 50 60 7
ng SA


16000-
14000-
c 12000-
E 10000-
8000-
o 6000-
O
4000-
2000-


45000-
40000-
35000-
S30000-
W25000-
-
=20000-
o
o 15000-
10000-
500-o
5000-
1


10 20 30 40 50 60
ng SA


16000
14000
S12000
E 10000
S8000
o 6000
4000
2000
0


0 10 20


30 40 50 60 70
ng SA


14000
12000-
10000-
. 8000-
-

o 6000-
4000-
2000-
0'


-Low [SA]
-Medium [SA]
High [SA]





S


. '


R2 = 0.9688
R2 = 0.9979


0 10 20 30 40 50 60 70


ng SA



Figure I (see legend on next page)


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0 100 200 300 400 500 600 7(
ng SA


{




0
0
S


A 0.5
X( 0.4
S 0.3 0
S0.2 A
0.1
*


*
Se

--__


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Figure I (see previous page)
Standard curve optimization. (A) Effect of plant extract on SA-induced luminescence. SA standards were made with either
LB or sid2-2 plant extract as the solvent. (B) Non-linearity of the SA-response curve. Data points were fitted with linear (blue)
and third order polynomial (orange) best-fit lines. Note the lower R-squared value of the linear best-fit line. (C) A typical set of
best-fit standard curves based on SA standards. The low SA concentration curve (orange) was fitted to standards of 0.8, 1.6,
and 3.2 ng SA. The medium SA concentration curve (blue) was fitted to standards of 8, 16, 24, and 32 ng SA. The high SA con-
centration curve (green) was fitted to standards of 40, 48, 56, and 64 ng SA. (D) Diminishing response of the biosensor to
increasing SA concentrations. (E) Effect of biosensor culture density on SA-induced luminescence. Biosensor cultures of OD600
= 0.6-0.8 were also tested and exhibited lower response to SA than OD600 = 0.4, but were omitted for clarity, as were error
bars. Values indicate the average of three replicates with standard deviation (A-D only). Experiments were done three times
with similar results.


ODo00 = 0.4. Cultures with ODs higher than 0.4 were less
responsive (Figure lE), indicating that this is the opti-
mum density for the assay. SA-induced luminescence var-
ied somewhat between experiments (data not shown), so
new SA standards were prepared for each experiment.

Optimization ofAcinetobacter sp. ADPWH_lux-based SA
Measurement
In order to further examine the specificity of the biosen-
sor, we tested 12 substances similar in structure to SA, but
not examined in [28] for their ability to induce lumines-
cence in ADPWH_ ux. These compounds are known to be
present in plants, and/or accumulate during pathogen
infection. None of the tested substances induced lumines-
cence, even at high concentrations (Additional file 1). To
improve upon the method of Huang et al. [28], a more
rapid extraction protocol was tested. To extract many sam-
ples at once, we used a Genogrinder 2000 homogenizer to
grind tissue that had been frozen in liquid nitrogen and to
extract the samples in LB. Samples were centrifuged and
the crude extract collected, omitting sonication. As
described previously, the extract was mixed with biosen-
sor culture and luminescence was measured [28]. SA con-
tent of wild-type plants infected with Pseudomonas syringae
pv. maculicola (Psm) ES4326 measured by the modified
method (described here) was similar to that obtained
with the original method (3.50 + 0.89 and 3.1 0.73 |ig
SA/g FW, respectively), indicating that these changes did
not significantly affect accuracy. To further confirm the
accuracy of the assay, we measured SA from varying quan-
tities of Psm ES4326-infected tissue. SA content increased
linearly with tissue mass (R2 = 0.9777, Figure 2A), con-
firming accuracy and suggesting little tissue (as few as 2-
3 leaves) is needed to obtain reproducible results, allow-
ing SA to be measured from single Arabidopsis plants
without a completely destructive harvest. However, we
typically used 5-6 leaves from different plants for each
sample to minimize plant-to-plant variation.

SAG Measurement
Although free SA is the biologically active form of SA, ele-
vation of SAG concentration accompanies activation of


plant defenses [30]. Consequently, measurement of SAG
has been used for detecting alterations in SA metabolism
[21]. Therefore, we developed a method for measuring
SAG using the biosensor. SAG has previously been meas-
ured by treating a dried extract of SAG with B-glucosidase,
releasing SA and glucose. The free SA is then analyzed by
HPLC [15]. This involves several extraction steps, resulting
in significant loss of SA. Since the biosensor detects SA in
a complex mixture, we added B-glucosidase directly to the
crude extract in order to avoid purification. Inclusion of P-
glucosidase did not affect luminescence induced by free
SA in a cell-free solution (Additional file 2). In the original
biosensor-based protocol, SA was extracted in LB (pH
7.0). However, the optimum pH for P-glucosidase is 5.6
[31]. Enzymatic hydrolysis of purified SAG has been pre-
viously carried out in acetate buffer (0.1 M, pH 5.6) [30].
To determine whether LB or acetate buffer was better for
B-glucosidase hydrolysis of SAG, we added B-glucosidase
to crude extracts prepared with these two solutions. Addi-
tionally, we carried out acid hydrolysis of SAG [31]. Enzy-
matic hydrolysis of SAG in the acetate buffer extract
released significantly more SA than in the LB extract (Fig-
ure 2B). An enzyme concentration of 0.03 U/ul crude
extract was sufficient for maximum SAG hydrolysis for
Psm ES4326-treated leaves (Additional file 3). Acid
hydrolysis of SAG resulted in ~2-fold lower SA detection
than enzymatic hydrolysis (Figure 2B); so acid hydrolysis
was no longer employed. Free SA content from tissue
extracted with acetate buffer did not differ significantly
from tissue extracted with LB (data not shown). Thereaf-
ter, all crude extracts were prepared with acetate buffer,
allowing the quantification of free and conjugated SA
from a single sample. When SAG was measured in this
way from varying quantities of Psm ES4326-infected tis-
sue, SA+SAG content increased linearly with tissue mass
(R2 = 0.9926, Figure 2C).

Comparison of ADPWHlux and GCIMS Salicylic Acid
Quantification
In order to compare our method of SA and SAG quantifi-
cation with existing methods, we added known amounts
of SA to plant extracts and analyzed them with


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0 20 40 60 80 100 120 140
Tissue weight (mg)


LB Acetate 2M HCI
buffer



y = 1.7961x 7.1647
R2 = 0.9926 /f


0 20 40 60 80
Tissue weight


100 120 140
(mg)


Figure 2
Accuracy of ADPWHlux-based SA and SAG quanti-
fication. (A) SA measurement of varying Psm ES4326-
infected tissue mass. (B) Comparison of extraction solvents
for SA+SAG quantification. Psm ES4326-infected tissue was
extracted with the indicated solvent. SA+SAG content was
then determined as in Methods. (C) SA+SAG measurement
of varying Psm ES4326-infected tissue mass. SA and SA+SAG
measurements were done as described in Methods.


y = 0.2538x + 0.873
R2 = 0.9777


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ADPWHIux and a previously established GC/MS method
[27]. As shown in Figure 3A, the ADPWH_lux-based
method detected higher levels of SA than did the GC/MS
method, and the values reported by ADPWH_lux were
closer to the amount of SA added. Both methods esti-
mated values that increased linearly with increasing SA
content. When the SA and SA+SAG content of Psm
ES4326-infected wild type tissue was analyzed over time,
the biosensor again reported higher concentrations than
the GC/MS method. Both methods reported the highest
concentration of free SA at 12 hpi, and the highest concen-
tration of SA+SAG at 24 hpi (Figures 3A and 3B respec-
tively).

SA Accumulation in Wild Type and sid2
To demonstrate the efficacy ofADPWH_Iux, we measured
SA and SAG in untreated and Psm ES4326-infected sid2-2
and wild-type plants. Psm ES4326 infection induced less
SA and SAG accumulation in sid2-2 than in wild type (Fig-
ures 4A and 4B). After Psm ES4326 infection, in wild type,
SA+SAG content was approximately 10-fold higher than
SA content. This ratio is similar to those obtained in pre-
vious studies that used similar pathogen treatments [32-
37] (Table 1). Wild type accumulated approximately six-
fold more SA, and approximately 40-fold more SA+SAG
than sid2. However, in wild type we obtained values for SA
and SA+SAG that were significantly higher than those of
previous studies (Table 1).

Evaluation of ADPWH_lux-based SA Quantification
The data presented in Figures 3 and 4 and in Table 1 sug-
gest more SA may be detected using ADPWHIux than
with previous methods. One explanation is that the bio-
sensor is responding to something other than SA that is
present in the crude extract, resulting in artificially high
values. Although several compounds that are structurally
similar to SA and/or accumulate during the defense
response do not induce luminescence in ADPWHIux [28]
(Additional file 1), we cannot exclude this possibility.
Additionally, little luminescence was induced by patho-
gen-treated sid2 extracts, suggesting that if there is a com-
pound other than SA that induces ADPWH_Iux, it is not
present in sid2, and may be derived from isochorismate.
Another possibility is that recovery of SA using HPLC- and
GC/MS-based methods which include organic solvent
extraction and evaporation steps result in partial recovery
of SA [38], despite inclusion of internal standards to
account for the loss of SA. Although these internal stand-
ards have been shown to have similar recovery rates to SA
[38], a difference in SA recovery between methods cannot
be ruled out. Additionally, differences in photoperiod,
pathogen inoculum, and the time after inoculation when
SA content is measured, may also contribute to differences
in SA measurements across different studies. Another pos-
sible cause of differing results across methodologies is


200
<
un 150
+
<
U) 100
r-
50

n


Plant Methods 2008, 4:28


0







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70.0

60.0

50.0

40.0

30.0

20.0


10.0

0.0
0.0


4500-
4000-
3500-
3000-
2500-
2000-
1500-
1000-
500-
0


35000

30000-

25000-

20000-

15000-

10000-

5000-


10.0 20.0 30.0
ng SA added


EADPWHIux
1GC/MS


40.0 50.0 60.0


12 24


Hours post-innoculation


EADPWHlux
GGC/MS


01 I
0 6 12
Hours post-innoculation


Figure 3
Comparison of ADPWHIux- and GC/MS-based methods for SA quantification. (A) Quantification of SA from plant
extracts with known amounts of SA added. The same extracts were used for SA quantification with each method. (B) Free SA
from Psm ES4326-infected wild type. Known SA amounts added were 0.6, 2.2, 3.8, 8.6, 16.6, 32.6, and 48.6 ng. (C) SA+SAG
from Psm ES4326-infected wild type. Values are the mean of 8 samples read in triplicate with standard deviation.




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Table I: Comparison of SA quantification results


Reference

This Study

Lee et al., 2006 [32]

Ishikawa et al., 2006 [33]

Nandi et al., 2003 [34]

This Study

Zheng et al., 2007 [35]

Gupta et al., 2000 [36]

Glazebrook et al., 2003 [37]


Treatment

Untreated

Untreated


Dpi Photoperiod (hr)


SA (.tg/g FW) SA+SAG (.tg/g FW)


Untreated

Untreated

PsmES4326 OD600= 0.001

PsmES4326 OD00 = 0.0001

PsmES4326 OD600 = 0.002

PsmES4326 OD600 = 0.002


ND: Not detectable. A dash indicates SA was not determined.


methyl-SA accumulation, which induces luminescence in
the biosensor [29]. However, in Psm ES4326-infected wild
type, methyl-SA reached a maximum concentration of
only 65 ng/g FW during pathogen infection (data not
shown). Given this low value, it appears that methyl-SA
accumulation contributes minimally to estimates of SA
accumulation, and was therefore not included in the anal-
ysis.

Despite differences with existing methods in terms of
absolute SA concentration, the ADPWHlux-based SA
quantification is useful for comparing SA content in
response to mutation and pathogen treatment. The values
obtained for SA and SA+SAG was also highly reproduci-
ble. Consistently, free SA accumulation at 48 hpi was -3.5
jig SA/g FW and SA + SAG was ~40 jig SA/g FW. The bio-
sensor-based method routinely produced standard devia-
tions between 15% and 25% of the mean and had a
minimum detection limit of about 0.28 jig SA/g FW (data
not shown). HPLC-based methods report standard devia-
tions which are ~12% of the mean, and can vary in detec-
tion limit, depending on the protocol and
instrumentation used [38,39]. A schematic of the biosen-
sor-based methodology and a detailed protocol are pre-
sented in Figure 5 and Additional file 4, respectively. In
our laboratory, free and conjugated SA was routinely
quantified from ~50 samples in ~5.0 hr.

Conclusion
In this study we present an improved method for the
quantification of SA from plant tissue using the SA biosen-
sor Acinetobacter sp. ADPWHlux. The modified method is
as accurate and more rapid than the previous Acinetobacter
sp. ADPWH_lux -based approach [28]. We also developed


a biosensor-based method for measuring SA + SAG using
enzymatic hydrolysis. Free and conjugated SA can be
measured simultaneously from hundreds of samples per
day, providing an alternative to HPLC and GC/MS, with
significant reductions in cost and processing time. Adop-
tion of 96-well formats for tissue grinding, SA extraction,
and SAG hydrolysis will further decrease the cost and time
involved. It is our hope that this methodology will
encourage investigators to include SA quantification in
their experiments, facilitating a more thorough under-
standing of this intriguing molecule.

Methods
Preparation of crude extract
This procedure was adapted from Huang et al. [28]. SA
measurements were carried out as follows unless other-
wise indicated. On the day of SA measurement, samples
were frozen in liquid nitrogen and ground at 1500
strokes/min for 30 sec in a Genogrinder 2000 (BT&C/OPS
Diagnostics, Bridgewater, NJ). Tissue was ground three
times while refreezing in liquid nitrogen each time. After
the third round of grinding, samples were left at room
temperature for 5 minutes, and 2.5 lil/mg tissue of room
temperature acetate buffer (0.1 M, pH 5.6) was added.
Samples were then mixed for 1 min at 1000 strokes/min
and centrifuged for 15 min at 16,000 g. Half (100 il) of
the supernatant was stored on ice for free SA measurement
and half was incubated at 37 C for 90 min with 4 U of 3-
glucosidase (3.2.1.21, Sigma-Aldrich, St. Louis, MO) for
SAG measurement.







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5000
4500
4000
3500
3000

600

400


200.

0.
Psm ES4326


60000

50000

40000

30000

1500


1000.

500.

0-
Psm ES4326


Figure 4
SA measuremer
infected wild-tvy


sid2 Wild type sid2
sid2 Wild type sid2


Wild type
Wild type


using a Victor3 Perkin Ellmer Multi-Detection Microplate
Reader (PerkinElmer, Waltham, Massachusetts). Each
sample was measured in triplicate. GC/MS based analysis
of SA follows from Schmelz et al. [27]. Briefly, aliquots of
crude extracts described above where spiked with 100 ng
of 2H,-SA as an internal standard and mixed with 300 Al
of H20:1-propanol: HC1 (1:2:0.005) followed by 1 ml of
dichloromethane (MeCl2). The MeCl2:1-propanol layer
containing SA was then transferred to a glass vial and 2 tl
of 2.0 M trimethylsilyldiazomethane solution was added
to form methyl esters. Residual derivatization agent was
neutralized with excess acetic acid. Vapor phase extraction
at 200 C was used to recover the MeSA on filters contain-
ing 30 mg Super Q (Alltech Associates, Inc., Deerfield, IL,
USA) followed by elution with MeCl2. Samples were then
analyzed with an established isobutane chemical ioniza-
tion-GC/MS profiling method [27]. Estimates of salicylic
acid (SA) represent combined pools of endogenous free
acids and methyl esters.

Standard curve
Known amounts of SA were dissolved in either LB or ace-
tate buffer, then diluted 10-fold in plant extract. SA stand-
ards were read in parallel with the experimental samples.
Conversion of luminescence to SA concentration was
done as discussed in Results.

Competing interests
The authors declare that they have no competing interests.


Authors' contributions
CTD contributed to the conception and design of the
I project, collected, analyzed, and interpreted the data for
111, all biosensor-based SA measurements, and prepared the
+ + manuscript. EAS collected, analyzed, and interpreted the
data for all GC/MS-based SA measurements, and revised
sid2 Wild type sid2 Wild type and edited the manuscript. ZM was involved in the con-
ception and design of the project, revised and edited the
nt of untreated and Psm ES4326- manuscript, and is the PI of the laboratory. All authors
e and sid2-2 plants. (A) SA. (B) read and approved the final manuscript.


SA+SAG. Values are the mean of 8 samples read in triplicate
with standard deviation. Experiments were done three times
with similar results.


Detection of salicylic acid using Acinetobacter sp.
ADPWH lux and GCIMS
An overnight culture of Acinetobacter sp. ADPWH_Iux was
diluted in 37C LB (1:20) and grown for ~3 hrs at 200
rpm to an OD600 of 0.4. Twenty Al of room temperature
crude extract was added to 60 il room temperature LB in
a black 96-well black cell culture plate. Using a multipi-
pet, 50 il of biosensor culture was added to each well and
mixed by pipet action. The plate was incubated at 37C
for 1 hr without shaking before luminescence was read


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1) Place 100 mg leaf tissue and
one metal bead in a tube and
freeze in liquid nitrogen.

2) Grind in Genogrinder 2000 or
equivalent homogenizer for
30 seconds.




3) Add 250 pl acetate buffer --
acetate buffer.


4) Extract in Genogrinder 2000
or equivalent homogenizer for
1 minute then centrifuge at
16,000 g for 15 min. i




5) Transfer the crude extract
into two new tubes.


6) Incubate one tube with 4 -glucosidase
P-glucosidase at 370C for 1.5 37C
hr, while storing the other tube
on ice. Then centrifuge all
samples for 5 min at 16,000 g.

7) Add each of the following to
wells in a microplate:

20 pl of crude extract
60 pl LB
50 pl Acinetobacter sp. OOOOOOOOOO00
ADPWHlux culture 000000000000
000000000000
Incubate 1 hr at 370C, then OOO000000000000
read luminescence. 000000000000
000000000000


Figure 5
Schematic ofAcinetobacter sp. ADPWH_Iux-based SA and SAG quantification.



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Additional material


Additional file 1
Specificity of ADPWH_ ux. The indicated compounds were added to
ADPWH lux and luminescence determined as described in Methods.
Values are the mean of 4 samples read in triplicate with standard devia-
tion. This experiment was done twice with similar results.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1746-
4811-4-28-Sl.jpeg]

Additional file 2
I ith .f fP-glucosidase on free SA detection byADPWHlux. f-glucosi-
dase was added to plant extract containing known amounts of SA, and
luminescence was determined with ADPWH lux as described in Meth-
ods. Values are the mean of 4 samples read in triplicate with standard
deviation. This experiment was done twice with similar results.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1746-
4811-4-28-S2.jpeg]

Additional file 3
Determination of the minimum effective quantity of P-glucosidase for
the determination of SA+SAG. f-glucosidase was added to Psm
ES4326-treated plant extract in increasing amounts and SA+SAG was
determined as described in Methods.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1746-
4811 -1 _- i..--]

Additional file 4
Protocol. Detailed protocol for Acinetobacter sp. ADPWHlux-based
SA measurement.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1746-
4811-4-28-S4.doc]




Acknowledgements
We thank Dr. lan Blaby (University of Florida, FL) for critical reading of the
manuscript, Dr. Hui Wang (NERC/Centre for Ecology and Hydrology-
Oxford, Oxford, UK) for the SA biosensor strain Acinetobacter sp.
ADPWH_lux and technical assistance with the SA measurement, and Drs.
Max Teplitski (University of Florida, FL) for access to the Victor3 Perkin
Ellmer Multi-Detection Microplate Reader and critical reading of the man-
uscript. This work was supported by a grant from the Herman Frasch Foun-
dation for Chemical Research and a research innovation grant from the
Institute of Food and Agricultural Sciences, University of Florida awarded
to ZM. CD was supported by an Alumni Fellowship from the University of
Florida.

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