Group Title: Plant Methods
Title: A high-throughput method for isolation of salicylic acid metabolic mutants
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 Material Information
Title: A high-throughput method for isolation of salicylic acid metabolic mutants
Series Title: Plant Methods
Physical Description: Archival
Creator: George Marek
Ryan Carver
Yezhang Ding
Deepak Sathyanarayan
Xudong Zhang
Zhonglin Mou
Publisher: BioMed Central
Publication Date: 2010
 Notes
Abstract: BACKGROUND: Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants. RESULTS: On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, npr1 (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six npr1 suppressors that accumulated lower levels of SA than npr1 upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 and their identity merits further investigation. CONCLUSIONS: The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor Acinetobacter sp. ADPWH_lux-based SA estimation, and allowed the screening for npr1 suppressors that accumulated less SA than npr1 after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.
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Marek et al. Plant Methods 2010, 6:21
http://www.plantmethods.com/content/6/1/21


PLANT METHODS


A high-throughput method for isolation of

salicylic acid metabolic mutants

George Marekt, Ryan Carvert, Yezhang Ding, Deepak Sathyanarayan, Xudong Zhang, Zhonglin Mou*


Abstract
Background: Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants.
Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC
and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We
recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_/ux based SA quantification,
which enables high-throughput analysis. In this study we describe an improved method for fast sample
preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.
Results: On the basis of the previously described biosensor-based method, we simplified the tissue collection and
the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA
was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and
centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen
infected wild-type, nprl (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as
did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of
the protocol presented here. We adapted this protocol to a high-throughput format and identified six nprl
suppressors that accumulated lower levels of SA than nprl upon pathogen infection. Two of the suppressors were
found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than nprl to the
bacterial pathogen Pseudomonas syringe pv. maculicola ES4326 and their identity merits further investigation.
Conclusions: The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time
required for the biosensor Acinetobacter sp. ADPWH_/ux based SA estimation, and allowed the screening for nprl
suppressors that accumulated less SA than nprl after pathogen infection in a high-throughput manner. The highly
efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization
of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in
the SA-related signaling pathways.


Background
Salicylic acid (SA) is a key signaling molecule in plant
defense against biotrophic pathogens [1,2]. Upon patho-
gen attack, SA accumulates in plant cells [3,4]. Preven-
tion of SA accumulation leads to disease susceptibility
[5], whereas treatment with SA confers resistance to a
variety of biotrophic pathogens [6,7]. Thus, understand-
ing the mechanisms underlying SA accumulation is
critical in the study of plant immunity.


* Correspondence zhlmou@ufledu
t Contributed equally
Department of Microbiology and Cell Science, University of Florida, P O Box
110700, Gainesville, FL, 32611, USA


0 BioMed Central


Nawrath and M6traux [8] performed a genetic screen
for Arabidopsis mutants that do not accumulate SA
after pathogen infection and identified two genetic loci,
SID1/EDSS and SID2/EDS16, which were later shown to
encode a chloroplast MATE (multidrug and toxin extru-
sion) transporter [9] and an SA biosynthetic enzyme
ICS1 (isochorismate synthase) [10], respectively. In the
screen, Nawrath and M6traux used an HPLC-based
method to quantify the SA levels in the pathogen-
infected leaf tissues from about 4,500 individual M2
plants. Because the HPLC-based method involves
extraction of SA in organic solvents, evaporation of
organic solvents, chromatographic purification and
detection by fluorescence spectroscopy [11,12], it is


2010 Marek et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution Licene (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.






Marek et al. Plant Methods 2010, 6:21
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extremely costly and time-consuming. To screen for
more SA metabolic mutants, a much faster and less
expensive method is needed.
Huang et al. recently developed an SA biosensor,
named Acinetobacter sp. ADPWH_lux [13]. This strain
is derived from Acinetobacter sp. ADP1, and contains a
chromosomal integration of a salicylate-inducible lux-
CDABE operon. The operon encodes a luciferase (LuxA
and LuxB) and the enzymes that produce its substrate
(LuxC, LuxD and LuxE) so cells that express the cluster
emit the 490-nm light spontaneously [14]. The biosen-
sor is highly specific to SA, methyl-SA, and the synthetic
SA derivative acetylsalicylic acid [13], thus suitable for
the quantification of SA from crude plant extracts.
We previously described an approach for the simulta-
neous quantification of free and glucose conjugated SA
from Arabidopsis leaf extracts using Acinetobacter
sp. ADPWH_lux [15]. Here we present a further shor-
tened protocol for the estimation of SA levels in patho-
gen-infected leaf tissue. Using the protocol described,
we have performed a genetic screen for suppressors of
the nprl (nonexpressor of pathogenesis-related genes)
mutant that hyperaccumulates SA during pathogen
infection [16,17].

Results
Rapid Extraction of SA by Direct Boiling of Leaf Discs
The method we previously described comprises leaf tis-
sue collection (weighing samples), grinding, extraction
in LB or acetate buffer, and centrifugation [15]. The
resulting crude leaf extract is then mixed with a culture
of the SA biosensor in a 96-well cell culture plate, and
incubated at 37C for one hour. The luminescence is
then determined. Compared with the conventional
HPLC or GC/MS method [11,18], the biosensor-based
method is much faster and requires little tissue (as few
as 2-3 leaves) [15]. However, the tissue collection (espe-
cially weighing samples) and the extraction procedure
are still time-consuming. To search for an even faster
method, we collected leaf discs (0.7 cm in diameter,
omitting weighing the samples) with a hole punch from
the bacterial pathogen Pseudomonas syringae pv. macu-
licola (Psm) ES4326-infected leaves of wild-type Col-0,
nprl and sid2 plants. We used pathogen-infected Col-0,
nprl and sid2 plants as samples because they contain
significantly different levels of SA [8,17]. Each leaf disc
was placed in 200 pL of LB in a 1.5-mL eppendorf tube,
and boiled at 95'C for 20 minutes. The LB extracts were
cooled down to room temperature, and then mixed with
the SA biosensor and incubated at 37C for one hour.
The luminescence was then determined. We found that
SA was readily extracted by the direct boiling of leaf
discs. As shown in Figure 1A, the pathogen-infected
Col-0 and nprl samples had significantly higher


. 400000
E
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8 200000

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E

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8
< 50000

al
u- 0


t

Untreated Psm ES4326

















Untreated Psm ES4326


10
u. 8-

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o C7 V- D C7 I-
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Untreated Psm ES4326
Figure 1 The direct boiling method in comparison with
the previously described biosensor- and HPLC-based methods.
(A) Luminescence from Psm ES4326-infected Col-, nprl and sid2
detected by the direct method. (B) Luminescence from Psm
ES4326-infected Col-0, nprl and sid2 detected by the previously
described biosensor-based method. (C) Free SA levels in Psm
ES4326-infected Col-0, npr and sid2 detected by the HPLC-based
method. Values are te mean of 8 samples (A), 6 samples read in
triplicate (B), and 4 samples (C) with standard deviation.


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luminescence than uninfected samples, whereas the sid2
samples just had background luminescence regardless of
whether they were infected or not. Furthermore, the
luminescence emitted by the infected nprl samples was
significantly stronger than that emitted by the infected
Col-0 samples (Figure 1A). The differences among Col-
0, nprl and sid2 were very similar to those revealed by
the previously described biosensor-based method and
the HPLC-based approach (Figures 1B and 1C). These
results suggest that the direct boiling method is able to
detect the differences in SA levels among different geno-
types, thus suitable for genetic screens for mutants with
altered SA levels.

High-Throughput Screening for SA Metabolic Mutants
We adapted the direct boiling protocol to a high-
throughput format competent for genetic screens. The
strategy is schematically described in Figure 2A and a
detailed protocol is presented in Additional file 1.
Again, we used Col-0, nprl and sid2 to test the efficacy
of the strategy. Seedlings of Col-0, nprl or sid2 were
transplanted into one third (32 pots) of a 96-pot tray


A
Growing plants in a 96-pot tray

Pathogen inoculation
(one leaflplant)

Sample collection
(one disc/leaf)
t
96-well PCR plate
(200 iL LB in each well)


Boiling at 95C for 20 min
in a 96-well PCR machine

96-well black culture plate
(50 iL of biosensor culture
in each well)


(Figure 2B). Three weeks later, half of the plants of each
genotype were inoculated with Psm ES4326 (Figure 2B).
To save the time spent on inoculation, only one leaf on
each plant was inoculated. Twenty-four hours later, a
leaf disc from each inoculated leaf was collected using a
hole punch, and placed into 200 pL of LB in a corre-
sponding well of a 96-well PCR plate. After all 96 leaf
discs (from the 96 plants in Figure 2B) were collected
and placed into the 96 wells, the PCR plate was put in a
PCR machine and heated at 95C for 20 minutes. The
extracts were cooled down to room temperature, and
50 pL of each extract was added into a corresponding
well in a black 96-well culture plate loaded with 50 tL
of a freshly prepared biosensor culture in each well.
After incubation at 37C for 1 hour in the dark, lumi-
nescence was assayed using a microplate luminometer.
As shown in Figure 2C, the differences in SA levels
among the three genotypes were clearly detected using
the "96-pot tray/96-well PCR plate/96-well culture
plate" format.
We then attempted to set up a mutant screen aimed
at identifying new components involved in regulating


B
Col-O sid2-1 nprl-3

PsmES4326 + + - + + - + + -


400000
300000
200000
100000
0


Psm ES4326 + + --+ + --+ +--

Col-O sid2-1 nprl3


Incubation Luminescence assay
at 37C for 1 hr using a microplate reader
Figure 2 High-throughput strategy for isolation of SA metabolic mutants. (A) Schematic of the "96-pot tray/96-well PCR plate/96-wel
culture plate" screen strategy. (B) Col-0, nprl and sid2 plants grown in a 96-cell tray treated with or without Psm ES4326. (C) Luminescence from
the Col-0, nprl and sid2 plants in (B) treated with or without Psm ES4326.


Page 3 of 7






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SA accumulation. Since the nprl mutant accumulates
significantly higher levels of SA than wild type during
pathogen infection, and NPR1 is a key positive regulator
of SA-mediated immune responses [19-21], we reasoned
that nprl suppressors, which accumulate less pathogen-
induced SA than nprl, would help uncover important
regulators of plant immunity. We therefore decided to
use the nprl mutant as starting material for the screen.
One gram of nprl seeds were treated with ethyl
methanesulfonate (EMS) and sown on soil. M2 seeds
were collected in pools when the M1 plants matured.
After germination, M2 seedlings were transplanted into
96-pot trays and screened as described in Figure 2A. So
far, approximately 10,000 M2 plants have been screened.
Figure 3 shows the luminescence from the plants in a
randomly selected tray in the primary screen. The pri-
mary screen has identified 35 putative nprl suppressors.
The putative suppressors were re-screened using the
direct boiling protocol. In the secondary screen, three
M3 plants from each putative suppressor were assayed.
As shown in Figure 4, six putative suppressors had
lower luminescence than nprl.

Confirmation of the Putative SA Metabolic Mutants Using
HPLC
To further confirm that the suppressors accumulate less
SA than nprl after pathogen infection, we measured SA
levels in the suppressors using HPLC. As shown in
Figure 5, after Psm ES4326 infection, all six suppressors
accumulated lower levels of both free and total SA than
nprl, suggesting that the suppressors contain mutations
that modified the SA accumulation pathway in nprl.
Interestingly, suppressors 62 and 69 accumulated very
low levels of SA, similar to the previously characterized
eds5 and sid2 mutant. To further characterize these two
suppressors, we measured Psm ES4326-induced SA
levels in the F, plants of the following crosses: 62 x 69,
62 x nprl, 69 x nprl, 62 x eds5, 62 x sid2, 69 x eds5,


'- 400000

49 300000-

0 200000

Cn 100000-

0)
0.

o '.

Figure 4 Luminescence from six putative nprl suppressors in
the secondary screen. Luminescence from Psm ES4326-infected
M3 plants of six putative nprl suppressors was determined using
the direct method.

and 69 x sid2. As shown in Figure 6, after Psm ES4326
infection, the Fi plants of 62 x 69 accumulated similar
levels of SA as 62 and 69, whereas the Fi plants of 62 x
nprl and 69 x nprl had similar amounts of SA as nprl,
suggesting that 62 and 69 are allelic and contain a reces-
sive mutation, since the 62 and 69 alleles will be hetero-
zygous in the F, Plants. Furthermore, the F, plants of
62 x eds5 and 69 x eds5 accumulated similar levels of
SA as eds5, whereas the Fi plants of 62 x sid2 and 69 x
sid2 had similar amounts of SA as wild type, indicating
that suppressors 62 and 69 are alleles of the eds5
mutant.
To identify the genetic mutations in 62 and 69, the
open reading frames of the two alleles of EDS5 were
sequenced. The allele 62 carries a transition mutation
converting a TGG to a premature stop condon (TGA)
at nucleotide 753 of the coding region, whereas the 69
mutation is caused by a G-to-A transition in the AG
from the splice acceptor site in intron 5, which may
lead to an abnormal splicing at the border of intron


Page 4 of 7


S1500000
1 nprl-3 plant and 95 M2 plants

o 1000000
UI

4 500000



t t t
nprl-3 Putative #1 Putative #2
Figure 3 Luminescence from a randomly picked tray of plants in the primary screen. Luminescence from Psm ES4326-infected M plants
was determined using the direct method in a "96-pot tray/96-well PCR plate/96-well culture plate" format. Arrows indicate the putative
nprl suppressors.






Marek et al. Plant Methods 2010, 6:21
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70 Ic


Figure 5 SA levels accumulated in the nprI suppressors
determined by HPLC. Free (A) and total (B) SA levels in Psm
ES4326-infected nprl suppressors were assayed using the HPLC-
based method. Values are the mean of 4 samples with standard
deviation. The experiment was repeated with similar results. The
suppressors 62 and 69 accumulated less SA than nprl
(*P < 0.0001).


5/exon 6. These results indicate that the mutant screen
identified two new alleles of the previously isolated eds5
mutants [9].

Pathogen Resistance Test for the SA Metabolic Mutants
To test whether the genetic mutations in the SA meta-
bolic mutants affect pathogen resistance, we inoculated
nprl-3 and the four mutants, 34, 46, 49, and 79, with
the bacterial pathogen Psm ES4326. The eds5 alleles, 62
and 69, were excluded since eds5 has been well charac-
terized [9]. The in plant growth of Psm ES4326 was
monitored three days after inoculation. As shown in Fig-
ure 7, Psm ES4326 grew significantly more in the SA
metabolic mutants than in nprl-3. The growth of Psm
ES4326 in 49 was 10-fold higher than in nprl-3. This
result is significant since nprl-3 is already highly


o cs -v- Mt t to N

oQ-t



80

U-
60-
0)
40-



0
"' I ,0 0 ii)p' 1
StI- ---- (0 (





Figure 6 SA levels accumulated in the F, plants determined by
HPLC. Free (A) and total (B) SA levels in Psm ES4326-infected Fa
plants of different crosses among 62, 69, nprl, eds5, and sid2 were
assayed using the HPLC-based method. Values are the mean of 4
samples with standard deviation. The experiment was repeated with
similar results.


susceptible to Psm ES4326 [16], demonstrating that the
high-throughput method developed in this study is valu-
able in identifying new components in the SA-mediated
defense signaling pathway.

Conclusions
Here we present a direct boiling protocol for the rapid
estimation of SA from plant tissue using the SA biosen-
sor Acinetobacter sp. ADPWH_lux. This protocol is
much faster and less expensive than the previously
described biosensor-based approaches [13,15]. The fast
sample preparation procedure, which comprises inocula-
tion of one leaf on each plant, collection of leaf discs, and
boiling in LB, significantly reduced the time spent on
inoculation, tissue collection, grinding and centrifugation.


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(0 0) 0
CV) RT IlN


Figure 7 Pathogen growth in the SA metabolic mutants. Leaves
of 4-week-old plants were inoculated with Psm ES4326 (OD,60 -
0.0001). The in plant bacterial titers were determined 3 days
postinoculation. Cfu, colony-forming units. Data represent the mean
of 8 independent samples with standard deviation. Psm ES4326
grew i I more in 34, 46, 49, and 79 than in nprl-3 (*P <
0.05, *P < 0.002). The experiment was repeated three times with
similar results.



This method was not designed to accurately determine
the SA concentration. Rather, it is intended to estimate
SA levels for rapid genetic screens with significant reduc-
tions in cost and processing time. We acknowledge that
SA levels induced by Psm ES4326 infection can vary
quite a lot among individuals of the same genotype,
depending on plant growth conditions. Additionally, this
method may not be sensitive enough in some applica-
tions, such as time-course quantifications of SA levels
with pathogen infection. However, the successful genetic
screen for suppressors of the nprl mutant has demon-
strated the efficacy of this high-throughput strategy. We
hope that the methodology presented in this study can
help saturate the genetic screens for SA metabolic
mutants, which in turn will facilitate a more thorough
understanding of this important plant defense molecule
[22,23].

Methods
Plant material and pathogen infection
The wild type used was the Arabidopsis thaliana (L.)
Heynh. Columbia (Col-0) ecotype, and the mutant
alleles used were nprl-3 [19], eds5-1 [9] and sid2-1 [8].
EMS mutagenesis was performed as described in [24].
Briefly, one gram of nprl-3 seeds were placed in 25 mL
of 0.2% EMS (v/v) in a 50-mL Falcon tube and incu-
bated on a rocking platform for 15 hours. After the
seeds were washed eight times with water, they were
suspended in 0.1% agarose and sown on soil. M2 seeds


were collected in pools when the M1 plants reached to
maturity. The M2 plants were germinated, transplanted
to 96-pot trays, and then grown at 22~25C under a 16
hr light/8 hr dark regime for three weeks. Infection of
plants with Psm ES4326 was performed as described
previously [25]. One leaf on each plant was infiltrated
with a suspension of Psm ES4326 (OD6oo = 0.001).

Preparation of crude extract
Twenty-four hours after Psm ES4326 infection, a leaf
disc was collected from each infected plant using a hole
punch and placed in 200 pl of LB in a well of a 96-well
PCR plate. The plate was then heated at 95C for 20
min in a PCR machine and cooled down to room
temperature.

Detection of salicylic acid using Acinetobacter sp.
ADPWH lux and HPLC
An overnight culture of Acinetobacter sp. ADPWH_lux
was diluted in 37C LB (1:20) and grown for ~2 hrs at
200 rpm to an OD600 of 0.4. Using a multipipette, 50 pl
of biosensor culture was added to each well in a 96-well
black cell culture plate, and then 50 pl of the crude
extract was added to each well and mixed by pipette
action. The plate was incubated at 37C for 1 hr with-
out shaking before luminescence was read using a Veri-
tas" Microplate Luminometer (Promega Corporation,
Sunnyvale, CA). Measurement of SA with HPLC was
done as described by Verberne et al. [11]. Briefly, -0.1
g tissues were ground in liquid nitrogen and extracted
with 1 mL of 90% methanol. After centrifugation at
14,000 g for 10 min, the supernatant was transferred
into a microcentrifuge tube. The pellet was extracted
with 0.5 mL of 100% methanol and the supernatant was
transferred to the same tube and dried in a speed
vacuum to final volume of -50 pL. The residue was
resuspended to 500 pL with hydrolysis buffer (0.1 M
sodium acetate buffer, pH 5.5). The mixture was equally
split into two microcentrifuge tubes [one for free SA,
the other for glucose conjugated SA salicylicc acid 2-0-
P-D-glucoside or SAG)]. For SAG, 10 units of 3-glucosi-
dase were added to the tube. After incubation at 37C
for 1.5 hr, an equal volume of 10% TCA was added to
both tubes. After centrifugation at 14,000 g for 10 min,
the supernatant was transferred to a fresh tube and par-
titioned with 1 mL extraction solvent (1 ethylacetate: 1
cyclohexane). The top organic phase was transferred to
a new tube, and dried in a speed vacuum to final
volume ~25 pL. The residue was resuspended to 0.25
mL with 0.2 M sodium acetate buffer (pH 5.5). After
centrifugation at 14,000 g for 10 min, the supernatant
was used for HPLC analysis. The sample was eluted
with 0.2 M sodium acetate buffer pH 5.5 in 10% metha-
nol at a flow-rate of 0.80 mL/min.


Page 6 of 7








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Statistical methods
All statistical analyses were performed with the data
analysis tools (t-TEST: Two Samples Assuming Unequal
Variances) in Microsoft Excel of Microsoft Office 2004
for Macintosh.


Additional material


Additional file 1: Detailed protocol for identification of SA
metabolic mutants using the direct boiling method




Acknowledgements
We thank Dr Hui Wang (NERC/Centre for Ecology and Hydrology-Oxford,
Oxford, UK) for the SA biosensor strain Acinetobacter sp ADPWH_/ux, and
Drs William Gurley and Sixue Chen (University of Florida, FL) for access to
the VeritasM Microplate Luminometer and the HPLC equipment, respectively
This work was supported by a grant from the Herman Frasch Foundation for
Chemical Research and a grant from the National Science Foundation (IOS
0842716) awarded to ZM, and publication of this article was funded in part
by the University of Florida Open-Access Publishing Fund

Authors' contributions
GM, RC, YD, DS, and XZ performed the experiments ZM designed the
project, wrote the manuscript, and is the PI of the laboratory All authors
read and approved the final manuscript

Competing interests
The authors declare that they have no competing interests

Received: 24 August 2010 Accepted: 23 September 2010
Published: 23 September 2010

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doi:10.1186/1746-4811-6-21
Cite this article as: Marek et al A high-throughput method for isolation
of salicylic acid metabolic mutants. Plant Methods 2010 6'21


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