Limits...
A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid.

Defraia CT, Schmelz EA, Mou Z - Plant Methods (2008)

Bottom Line: ADPWH_lux.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, P,O, Box 110700, Gainesville, FL, 32611, USA. zhlmou@ufl.edu.

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 (salicylic acid 2-O-beta-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.

Results: On the basis of the original biosensor-based method, we optimized extraction and quantification. SAG content was determined by treating crude extracts with beta-glucosidase, then measuring the released free SA with the biosensor. beta-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 mug SA/g FW. Samples typically had a standard deviation of up to 25% of the mean.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Comparison of ADPWH_lux- 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654556&req=5

Figure 3: Comparison of ADPWH_lux- 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.

Mentions: In order to compare our method of SA and SAG quantification with existing methods, we added known amounts of SA to plant extracts and analyzed them with ADPWH_lux 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 estimated 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 concentration of SA+SAG at 24 hpi (Figures 3A and 3B respectively).


A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid.

Defraia CT, Schmelz EA, Mou Z - Plant Methods (2008)

Comparison of ADPWH_lux- 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654556&req=5

Figure 3: Comparison of ADPWH_lux- 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.
Mentions: In order to compare our method of SA and SAG quantification with existing methods, we added known amounts of SA to plant extracts and analyzed them with ADPWH_lux 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 estimated 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 concentration of SA+SAG at 24 hpi (Figures 3A and 3B respectively).

Bottom Line: ADPWH_lux.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, P,O, Box 110700, Gainesville, FL, 32611, USA. zhlmou@ufl.edu.

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 (salicylic acid 2-O-beta-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.

Results: On the basis of the original biosensor-based method, we optimized extraction and quantification. SAG content was determined by treating crude extracts with beta-glucosidase, then measuring the released free SA with the biosensor. beta-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 mug SA/g FW. Samples typically had a standard deviation of up to 25% of the mean.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus