Presentation Title

Isolation and Identification of a Natural Product that Leads to Disease Resistance in Plant

Faculty Mentor

Dr. Gregory Barding

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 10:15 AM

Location

9-279

Session

Physical Sciences 1

Type of Presentation

Oral Talk

Subject Area

physical_mathematical_sciences

Abstract

Powdery mildew disease is a fungal infection which can have a negative effect on crop growth. There is evidence that a bio-signaling molecule is present due to a gene mutation in A. thaliana that results in the increase in resistance to the disease. By using metabolite standards as models for the molecule, a method for isolation and identification of the molecule is underway. Plant extracts containing the metabolite are prepared using C18 solid phase extraction, followed by weak anion exchange SPE. A bioassay is then used to confirm if the compound is present in the isolated fractions. The results of the bioassay have led to the hypothesis that the molecule is a highly polar, anionic compound. Further identification of the molecule will include the use of liquid chromatography fraction collection and high resolution NMR spectroscopy. The isolation and identification processes are being studied by using a variety of anionic and neutral metabolite standards to explore hydrophilic interaction liquid chromatography (HILIC). A successful method has been developed which allows for the separation of glucose, sucrose, glutamate, and galacturonic acid in a HPLC system. Changing the variables in the method provides insight regarding polar carbohydrate separation. Isolating and identifying the metabolite would provide information regarding the biological mechanism to the resistance of the fungal disease, which would have possible applications on the larger agricultural community.

Summary of research results to be presented

Using weak anion exchange solid phase extraction on plant extracts, the targeted analyte has been determined to be a highly anionic compound. The polarity and highly anionic characteristic of our unknown target has been confirmed through bioassays performed by our collaborators at UC Riverside. Because of these characteristics, isolation and identification of the compound will be exceedingly difficult. The most accessible option is to use hydrophilic interaction liquid chromatography (HILIC) due to its compatibility with volatile solvents and minimal additives. To this end, a HILIC column is currently being used to perform fraction collections of the plant extracts with different parameters. These collections are then tested with the bioassay to determine whether the parameters of the collection lead to collection of the targeted analyte. The first trial involved acidic aqueous and organic buffers. Acetic acid was used to make both of the buffers, however, residual acetic acid led to inconclusive bioassay results due to plant sensitivity to acetic acid, even under buffered conditions. Currently, acidic fraction collections are still being explored with the use of formic acid. A different approach is to use a slightly basic elution protocol using ammonium bicarbonate. Preliminary results suggest that not only can ammonium bicarbonate be removed through sublimation under vacuum, but is effective at eluting the targeted analyte. Currently, a method is under development to isolate the unknown analyte for identification.

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Nov 18th, 10:00 AM Nov 18th, 10:15 AM

Isolation and Identification of a Natural Product that Leads to Disease Resistance in Plant

9-279

Powdery mildew disease is a fungal infection which can have a negative effect on crop growth. There is evidence that a bio-signaling molecule is present due to a gene mutation in A. thaliana that results in the increase in resistance to the disease. By using metabolite standards as models for the molecule, a method for isolation and identification of the molecule is underway. Plant extracts containing the metabolite are prepared using C18 solid phase extraction, followed by weak anion exchange SPE. A bioassay is then used to confirm if the compound is present in the isolated fractions. The results of the bioassay have led to the hypothesis that the molecule is a highly polar, anionic compound. Further identification of the molecule will include the use of liquid chromatography fraction collection and high resolution NMR spectroscopy. The isolation and identification processes are being studied by using a variety of anionic and neutral metabolite standards to explore hydrophilic interaction liquid chromatography (HILIC). A successful method has been developed which allows for the separation of glucose, sucrose, glutamate, and galacturonic acid in a HPLC system. Changing the variables in the method provides insight regarding polar carbohydrate separation. Isolating and identifying the metabolite would provide information regarding the biological mechanism to the resistance of the fungal disease, which would have possible applications on the larger agricultural community.