Genetics of root exudates in Arabidopsis thaliana

Aparna Digamber Deshpande, Purdue University

Abstract

At any given time, plant roots release a distinct set of metabolites into the rhizosphere. The spectrum of exuded metabolites is influenced by environmental factors and by the plant's gene repertoire. The objectives of this project were to develop a profiling approach to detect a large number of metabolites in Arabidopsis root exudates, to analyze the effects of genotype on the accumulation of a subset of these exuded metabolites, and to determine the identity of these metabolites. Thin layer chromatography was found to be relatively insensitive, while derivatization efficiency limited the number of metabolites detected by gas chromatography. Reverse-phase high performance liquid chromatography-photodiode array detection was found to be adept at metabolically profiling Arabidopsis root exudates. Six UV-absorbent compounds were reproducibly found in root exudate profiles at 360 nm but were absent from seed exudates, indicating that they are released by living roots. Root exudate composition analyzed at 360 nm was highly similar or identical among five ecotypes of Arabidopsis thaliana, suggesting conservation of the metabolic pathways that yield these compounds. Screening 2000 M3 mutagenized families did not yield any detected mutations in the accumulation of the six major metabolites seen at 360 nm, suggesting that the six metabolites are synthesized from redundant pathways or are essential for plant viability. The accumulation of the six compounds in root exudates was found to be light-independent and their synthesis was found to be independent of chalcone synthase, chalcone isomerase, dihydroflavanol 4-reductase, favonoid 3'-hydroxylase, and ferulate 5-hydroxylase activities. Fourteen amino acids in un-hydrolyzed Arabidopsis root exudates were present in varying concentrations. Direct injection mass spectrometry of total root exudates detected several large ions but did not permit identification of compounds in these peaks. Structural analysis of the six major exudate compounds observed at 360 nm was attempted by LC-ESI-MS. Mass-to-charge ratio profiles of the six metabolites did not match any compounds previously identified in Arabidopsis. Characterized mutants yielded wild-type root exudate profiles at 360 nm. Hence, the six metabolites are likely novel compounds. Precise and accurate identification of these metabolites will require additional targeted analysis and comparison to standards.

Degree

Ph.D.

Advisors

Bennetzen, Purdue University.

Subject Area

Biochemistry

COinS