Dataset Information

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

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.

SUBMITTER: Defraia CT

PROVIDER: S-EPMC2654556 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Defraia Christopher T CT Schmelz Eric A EA Mou Zhonglin Z

Plant methods 20081231

<h4>Background</h4>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 a ...[more]

PMID: 19117519

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Project description:The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

Project description:BackgroundSalicylic 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.ResultsOn 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.ConclusionsThe 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.

Dataset Information

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

Background

Results

Conclusion

Publications

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

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