Page 246 - PC2019 Program & Proceedings

PLANT CANADA 2019

P65. Distinct metabolic modes drive variation in cyclic and acyclic monoterpenoid biosynthesis in
Pelargonium graveolens chemotypes
*
Bergman, M. ; M. Phillips
University of Toronto – Mississauga

Pelargonium (scented geraniums) is a genus of flowering plant in the Geraniaceae known for its pleasing
aromas. Its essential oils are used for fragrance and flavoring but also possess arachnicidal and
antimicrobial properties. Despite its widespread use in the cosmetics and cleaning industries, little is
known about Pelargonium essential oil biosynthesis. Here we demonstrate the contribution of at least two
distinct metabolic pathways responsible for the characteristic monoterpenoid volatile blend in
Pelargonium. The first group consists of the cyclic p-menthane monoterpenes (-)-isomenthone and (+)-
limonene which resemble high value monoterpenes found in peppermint but with inverted
stereochemistry. The second group, referred to here as citronelloid monoterpenes, include acyclic
monoterpene alcohols such as geraniol and (-)-citronellol, and their ester and aldehyde derivatives. Using
untargeted volatile profiling of 22 seed-grown lines of wild-type P. graveolens we identified 3 distinct
chemotypes which predominantly accumulate either (-)-isomenthone, geraniol, or (-)-citronellol with
minor contributions from approximately 80 other volatile compounds. We exploited the metabolic
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differences of these chemotypes in whole plant CO2 isotopic labelling assays to determine that (1) the p-
menthane monoterpenoids are likely synthesized from (+)-limonene via (+)-piperitone , (2) these two
groups of monoterpenes utilize a common pool of geranyl diphosphate (GDP) precursor supplied by the
2C-methyl-D-erythritol-4-phosphate pathway and (3) downstream of GDP, these two pathways are
functionally independent and do not appear to share common intermediates.

Matthew Bergman (matthew.bergman@mail.utoronto.ca)

P66. Mapping metabolic carbon partitioning in Arabidopsis rosette tissue using CO2 labeling and
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ammonia chemical ionization mass spectrometry
Phillips, M.; B. Davis *
University of Toronto – Mississauga

Flux studies in Arabidopsis have been facilitated by CO2 whole plant labeling techniques, but
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calculation of percent atom labeling across broad classes of metabolites remains a formidable task due to
the fragmentation inherent in electron impact GCMS analysis. Here we present a soft chemical ionization
(CI) approach to analyzing label incorporation into primary metabolites of Arabidopsis rosette tissue.
Compared to other CI reagent gases, ammonia features the highest proton affinity, resulting in little to no
fragmentation of analytes during ionization. We exploited this property to simplify the calculation of label
incorporation by preserving the intact molecular ion cluster containing unlabeled (M+0) and labeled
isotopologs (M+n) for any central metabolite detectable in a standard GCMS metabolomics strategy. We
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then subjected the same samples to isotope ratio mass spectrometry for an unbiased measure of total C
label assimilated by each plant during each labeling experiment. In this fashion, we were able to establish
the absolute commitment of freshly fixed carbon to major pathways of central metabolism. For instance,
we determined that while total carbon assimilation in plants increased with increasing light intensity, the
fraction of the total fixed carbon pool committed to sucrose biosynthesis remained constant (~19%).
Meanwhile, the methylerythritol-4-phosphate pathway received only about 1/3% of the plant’s total
carbon budget. This technique provides a quantitative basis for assessing metabolic engineering efforts to
reprogram plant metabolism.

Benjamin Davis (bd13md@brocku.ca)

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