In vivo 13C stable isotope tracing of single leaf development in the cold

Measuring the metabolite profile of plants can be a strong phenotyping tool, but the changes of metabolite pool sizes are often difficult to interpret, not least because metabolite pool sizes may stay constant while carbon flows are altered and vice versa. Hence, measuring the carbon allocation of metabolites enables a better understanding of the metabolic phenotype. The main challenge of such measurements is the in vivo integration of a stable or radioactive label into a plant without perturbation of the system. To follow the carbon flow of a precursor metabolite, a method is developed in this work that is based on metabolite profiling of primary metabolites measured with a mass spectrometer preceded by a gas chromatograph (Wagner et al. 2003; Erban et al. 2007; Dethloff et al. submitted). This method generates stable isotope profiling data, besides conventional metabolite profiling data. In order to allow the feeding of a 13C sucrose solution into the plant, a petiole and a hypocotyl feeding assay are developed. To enable the processing of large numbers of single leaf samples, their preparation and extraction are simplified and optimised. The metabolite profiles of primary metabolites are measured, and a simple relative calculation is done to gain information on carbon allocation from 13C sucrose. This method is tested examining single leaves of one rosette in different developmental stages, both metabolically and regarding carbon allocation from 13C sucrose. It is revealed that some metabolite pool sizes and 13C pools are tightly associated to relative leaf growth, i.e. to the developmental stage of the leaf. Fumaric acid turns out to be the most interesting candidate for further studies because pool size and 13C pool diverge considerably. In addition, the analyses are also performed on plants grown in the cold, and the initial results show a different metabolite pool size pattern across single leaves of one Arabidopsis rosette, compared to the plants grown under normal temperatures. Lastly, in situ expression of REIL genes in the cold is examined using promotor-GUS plants. Initial results suggest that single leaf metabolite profiles of reil2 differ from those of the WT.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Potsdam

Year

2014

Author dc:creator

• Dethloff, Frederik

Contributors dc:contributor

• Willmitzer, Lothar

• stable isotope tracing
• metabolism
• sucrose
• carbon flow
• qualitative pathway interpretation

Statement dc:rights

• Creative Commons - Namensnennung, Nicht kommerziell, Weitergabe zu gleichen Bedingungen 3.0 Deutschland

Repository record source_url

https://publishup.uni-potsdam.de/frontdoor/index/index/docId/6803

OAI identifier oai:identifier

oai:kobv.de-opus4-uni-potsdam:6803