Chemistry and Biochemistry Department
Thesis Defense
Detection and quantitationof trehalose and other sugars inrice and Arabidopsis tissueusing GC-MS
Elizabeth Martinez
Monday, May 2, 2016
3:00 – 5:00 PM
Bldg. 4/2-314
ABSTRACT:
Trehalose, a disaccharide commonly found in many organisms, is an important sugar often associated with increased tolerance to a variety of stressors. Understanding how trehalose levels change in plants during abiotic stress will enhance our understanding of how plants cope with increasing stresses associated with climate change. For this study, trehalose sucrose, glucose and fructose, were quantified in submergence tolerant and intolerant rice varieties to better understand carbon metabolism during complete flooding and subsequent recovery. In plants, trehalose is present in exceedingly low quantities making it difficult to detect and quantify, especially due to its low relative abundance compared with sucrose, fructose and glucose. The analysis was done using Gas chromatography – mass spectrometry (GC-MS) equipped with a quadrupole detector, a powerful technique for the sensitive detection of gas-phase analytes. Because of the low concentration of trehalose present in plants, it was important to clean up the samples by removing anionic metabolites through weak anion exchange (WAX) solid phase extraction (SPE). For data analysis of metabolites were derivatized with a suitable reagent, such as MSTFA, followed by GC-MS experiments carried out using a full scan (60-600 m/z) to identify each sugar using library matching as well as an in-house library generated with standards, and then selected ion monitoring (SIM) was used to further reduce the background associated with unwanted signals by only detecting specific ions associated with each sugar. The ions chosen were 317, 319, 323, and 361, and used to quantify (fructose, glucose, 13C6 glucose, and trehalose) the specific ions to the sugars. In addition to rice, different variations of Arabidopsis thaliana were evaluated for sugar content using SIM to quantify trehalose and a full scan to quantify sucrose, glucose, and fructose. It was determined the LOD and LOQ for trehalose in the rice samples are 0.640 ng, and 1.01 ng of trehalose per g of tissue, respectively, The LOD and LOQ for trehalose in the Arabidopsis samples are 0.744 ng, and 0.825 ng, respectively. This resulted in an average concentration of trehalose in M202 to be 11.5 ng of trehalose per g of tissue, in IR64 to be 1.3 ng of trehalose per g of tissue, and in Arabidopsis thaliana to be 17.9 μg of trehalose per g of tissue.
Committee members
Dr. Greg Barding (chair)
Dr. Lisa Alex
Dr. Yan Liu