Large-scale profiling of diterpenoid glycosides from Stevia rebaudiana using ultrahigh performance liquid chromatography/tandem mass spectrometry

Abstract

The plant Stevia rebaudiana accumulates a suite of diterpenoid metabolites that are natural sweeteners finding increased use as sugar substitutes. To guide breeding of stevia plants that accumulate substances with desirable flavor in high yield, rapid and accurate methods are needed to profile these substances in plant populations. This report describes an 8-min ultrahigh performance liquid chromatography-tandem mass spectrometry method for separation and quantification of seven stevia glycosides including steviolbioside; stevioside; rebaudiosides A, B, and C; rubusoside; and dulcoside as well as aglycones steviol and isosteviol. This negative mode electrospray ionization/multiple reaction monitoring method yielded low limits of detection <1 ng/mL for steviol, 6 ng/mL for isosteviol, and <15 ng/mL for all stevia glycosides. Stevioside and Reb A, B, and C were quantified in more than 1,100 extracts from stevia leaves as part of a large-scale profiling exercise. Leaf tissue levels in this population spanned about two orders of magnitude for stevioside (2-125 mg/g dry weight), Reb A (2.5-164 mg/g), Reb B (0.5-50 mg/g), and Reb C (1.5-125 mg/g), but levels of individual metabolites exhibited independent variation. The wide spread of metabolite levels highlights the utility and importance of performing targeted metabolic profiling for large plant populations.

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

“…9, B-D. Fragmentation of the oligogalactolipids resulted in expected peaks, including free fatty acids, lysolipids, and three forms in which both acyl tails were lost. In these forms, the headgroup remained attached to a version of the glycerol backbone, based on previous analyses of galactolipid fragmentation (83). Together with previous studies, these data confirm that the products are as suggested and that SFR2 is processive.…”

Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

“…Hence, steviolbioside was considered as an intermediate and not the rubusoside [31]. But isolation of rubusoside from Stevia has now been reported that suggests the possible involvement of this compound in steviol glycoside biosynthesis [33]. Additionally, rubusoside has been considered superior to stevioside because latter possessed a bitter after-taste [34].…”

Daily Light Integral Influences Steviol Glycoside Biosynthesis and Relative Abundance of Specific Glycosides in Stevia

“…The supernatant was transferred to a new 1.5-mL centrifuge tube. Extract supernatant was prepared for analysis by using a Millipore deep well filter plate and deep well 96-well receiving plate (Millipore Corporation, Billerica, MA) as previously described by Shafii et al (2012). Stevioside and rebaudiosides A, B, and C were quantified by ultra-high performance liquid chromatography-tandem mass spectrometry as previously described (Shafii et al, 2012) with the addition of rebaudioside D quantification with the following parameters: multiple reaction monitoring (MRM) transition m/z 1127 > 803, retention time 2.6 min, declustering potential -35 V, entrance potential -10 V, and collision potential -45 V. Morphological data collection.…”

“…Extract supernatant was prepared for analysis by using a Millipore deep well filter plate and deep well 96-well receiving plate (Millipore Corporation, Billerica, MA) as previously described by Shafii et al (2012). Stevioside and rebaudiosides A, B, and C were quantified by ultra-high performance liquid chromatography-tandem mass spectrometry as previously described (Shafii et al, 2012) with the addition of rebaudioside D quantification with the following parameters: multiple reaction monitoring (MRM) transition m/z 1127 > 803, retention time 2.6 min, declustering potential -35 V, entrance potential -10 V, and collision potential -45 V. Morphological data collection. At the time of tissue collection for glycoside analysis, the number of nodes on the tallest branch (opposite and alternate nodes were each counted as one), canopy height (cm, from soil line to tallest point), and average leaf area (one leaf measured twice collected from the seventh node down from the apex of the tallest branch) were determined.…”