A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid
© DeFraia et al; licensee BioMed Central Ltd. 2008
Received: 21 July 2008
Accepted: 31 December 2008
Published: 31 December 2008
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 (salicylic acid 2-O-β-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_lux. 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.
On the basis of the original biosensor-based method, we optimized extraction and quantification. SAG content was determined by treating crude extracts with β-glucosidase, then measuring the released free SA with the biosensor. β-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 μg SA/g FW. Samples typically had a standard deviation of up to 25% of the mean.
The ability of Acinetobacter sp. ADPWH_lux 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.
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 biotrophic pathogens [5, 8], and pathogen infection causes the accumulation of SA [9, 10]. SA can be glucosylated to form SAG (2-O-β-D-glucosylsalicylic acid), which serves as a biologically inert reservoir of SA . SA is also present in plants as methyl-salicylate, which can also be conjugated to glucose . Generally, mutants with constitutively high SA levels are resistant to biotrophic pathogens, 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 solvents and subsequent evaporation. SA is then purified chromatographically, and detected by fluorescence spectroscopy or mass spectrometry. However, during extraction 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 . This strain is derived from Acinetobacter sp. ADP1, and contains a chromosomal 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 . Measurement of SA from TMV-infected tobacco leaves with the biosensor and GC/MS yielded similar results , demonstrating that this strain is suitable for the quantification of SA from plant tissue.
Herein, we present an improved approach for the quantification of free SA from Arabidopsis leaf extracts using Acinetobacter sp. ADPWH_lux. We have also developed a method for Acinetobacter sp. ADPWH_lux-based SAG measurement.
Standard Curve Generation
[SA] = [(luminesence - y-interceptstandard curve)/slopestandard curve]/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 determine the SA concentration of samples with largely different luminescence values. We found this approach to be useful in determining SA content between 1.6 and 64 ng SA (0.28 and 11 μg SA/g FW). At higher concentrations, induction of the biosensor by SA was diminished (Figure 1D). If sample SA concentrations exceeded 11 μg 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 luminescence. The responsiveness of Acinetobacter sp. ADPWH_lux increased with culture density, reaching a maximum at OD600 = 0.4. Cultures with ODs higher than 0.4 were less responsive (Figure 1E), indicating that this is the optimum density for the assay. SA-induced luminescence varied somewhat between experiments (data not shown), so new SA standards were prepared for each experiment.
Optimization of Acinetobacter sp. ADPWH_lux-based SA Measurement
Although free SA is the biologically active form of SA, elevation of SAG concentration accompanies activation of plant defenses . Consequently, measurement of SAG has been used for detecting alterations in SA metabolism . Therefore, we developed a method for measuring SAG using the biosensor. SAG has previously been measured by treating a dried extract of SAG with β-glucosidase, releasing SA and glucose. The free SA is then analyzed by HPLC . This involves several extraction steps, resulting in significant loss of SA. Since the biosensor detects SA in a complex mixture, we added β-glucosidase directly to the crude extract in order to avoid purification. Inclusion of β-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 β-glucosidase is 5.6 . Enzymatic hydrolysis of purified SAG has been previously carried out in acetate buffer (0.1 M, pH 5.6) . To determine whether LB or acetate buffer was better for β-glucosidase hydrolysis of SAG, we added β-glucosidase to crude extracts prepared with these two solutions. Additionally, we carried out acid hydrolysis of SAG . Enzymatic hydrolysis of SAG in the acetate buffer extract released significantly more SA than in the LB extract (Figure 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). Thereafter, 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 tissue, SA+SAG content increased linearly with tissue mass (R2 = 0.9926, Figure 2C).
Comparison of ADPWH_lux and GC/MS Salicylic Acid Quantification
SA Accumulation in Wild Type and sid2
Comparison of SA quantification results
SA (μg/g FW)
SA+SAG (μg/g FW)
Lee et al., 2006 
Ishikawa et al., 2006 
Nandi et al., 2003 
Psm ES4326 OD600 = 0.001
Zheng et al., 2007 
Psm ES4326 OD600 = 0.0001
Gupta et al., 2000 
Psm ES4326 OD600 = 0.002
Glazebrook et al., 2003 
Psm ES4326 OD600 = 0.002
Evaluation of ADPWH_lux-based SA Quantification
The data presented in Figures 3 and 4 and in Table 1 suggest more SA may be detected using ADPWH_lux than with previous methods. One explanation is that the biosensor 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 ADPWH_lux  (Additional file 1), we cannot exclude this possibility. Additionally, little luminescence was induced by pathogen-treated sid2 extracts, suggesting that if there is a compound other than SA that induces ADPWH_lux, 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 , despite inclusion of internal standards to account for the loss of SA. Although these internal standards have been shown to have similar recovery rates to SA , 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 possible cause of differing results across methodologies is methyl-SA accumulation, which induces luminescence in the biosensor . 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 analysis.
In this study we present an improved method for the quantification of SA from plant tissue using the SA biosensor Acinetobacter sp. ADPWH_lux. The modified method is as accurate and more rapid than the previous Acinetobacter sp. ADPWH_lux -based approach . 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. Adoption 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 understanding of this intriguing molecule.
Preparation of crude extract
This procedure was adapted from Huang et al. . SA measurements were carried out as follows unless otherwise 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 μl/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 μl) 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 β-glucosidase (126.96.36.199, Sigma-Aldrich, St. Louis, MO) for SAG measurement.
Detection of salicylic acid using Acinetobacter sp. ADPWH_lux and GC/MS
An overnight culture of Acinetobacter sp. ADPWH_lux was diluted in 37°C LB (1:20) and grown for ~3 hrs at 200 rpm to an OD600 of 0.4. Twenty μl of room temperature crude extract was added to 60 μl room temperature LB in a black 96-well black cell culture plate. Using a multipipet, 50 μl of biosensor culture was added to each well and mixed by pipet action. The plate was incubated at 37°C for 1 hr without shaking before luminescence was read 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. . Briefly, aliquots of crude extracts described above where spiked with 100 ng of 2H6-SA as an internal standard and mixed with 300 μl of H2O:1-propanol: HCl (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 μl 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 containing 30 mg Super Q (Alltech Associates, Inc., Deerfield, IL, USA) followed by elution with MeCl2. Samples were then analyzed with an established isobutane chemical ionization-GC/MS profiling method . Estimates of salicylic acid (SA) represent combined pools of endogenous free acids and methyl esters.
Known amounts of SA were dissolved in either LB or acetate buffer, then diluted 10-fold in plant extract. SA standards were read in parallel with the experimental samples. Conversion of luminescence to SA concentration was done as discussed in Results.
We thank Dr. Ian 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 manuscript. This work was supported by a grant from the Herman Frasch Foundation 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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