title: A fast and simple LC-MS-based characterization of the flavonoid biosynthesis pathway for few seed(ling)s.
abstract: (Pro)anthocyanidins are synthesized by the flavonoid biosynthesis pathway with multi-layered regulatory control. Methods for the analysis of the flavonoid composition in plants are well established for different purposes. However, they typically compromise either on speed or on depth of analysis.
  
  
  
  In this work we combined and optimized different protocols to enable the analysis of the flavonoid biosynthesis pathway with as little as possible biological material. We chose core substances of this metabolic pathway that serve as a fingerprint to recognize alterations in the main branches of the pathway. We used a simplified sample preparation, two deuterated internal standards, a short and efficient LC separation, highly sensitive detection with tandem MS in multiple reaction monitoring (MRM) mode and hydrolytic release of the core substances to reduce complexity. The method was optimized for Arabidopsis thaliana seeds and seedlings. We demonstrate that one Col-0 seed/seedling is sufficient to obtain a fingerprint of the core substances of the flavonoid biosynthesis pathway. For comparative analysis of different genotypes, we suggest the use of 10 seed(lings). The analysis of Arabidopsis thaliana mutants affecting steps in the pathway revealed foreseen and unexpected alterations of the pathway. For example, HY5 was found to differentially regulate kaempferol in seeds vs. seedlings. Furthermore, our results suggest that COP1 is a master regulator of flavonoid biosynthesis in seedlings but not of flavonoid deposition in seeds.
  
  
  
  When sample numbers are high and the plant material is limited, this method effectively facilitates metabolic fingerprinting with one seed(ling), revealing shifts and differences in the pathway. Moreover the combination of extracted non-hydrolysed, extracted hydrolysed and non-extracted hydrolysed samples proved useful to deduce the class of derivative from which the individual flavonoids have been released.
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany.
authors: Benjamin
authors: B
authors: Jaegle
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany.
authors: Miran Kalle
authors: MK
authors: Uroic
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany.
authors: Xu
authors: X
authors: Holtkotte
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany.
authors: Christina
authors: C
authors: Lucas
authors: Department of Chemistry, University of Cologne, Greinstr. 4, 50939, Cologne, Germany.
authors: Andreas Ole
authors: AO
authors: Termath
authors: Department of Chemistry, University of Cologne, Greinstr. 4, 50939, Cologne, Germany.
authors: Hans-Günther
authors: HG
authors: Schmalz
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany.
authors: Marcel
authors: M
authors: Bucher
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany.
authors: Ute
authors: U
authors: Hoecker
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany. martin.huelskamp@uni-koeln.de.
authors: Martin
authors: M
authors: Hülskamp
authors: Botanical Institute and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne Biocenter, Zülpicher Str. 47b, 50674, Cologne, Germany. andrea.schrader@uni-koeln.de.
authors: Andrea
authors: A
authors: Schrader
mesh_terms: D000872:Anthocyanins / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D005419:Flavonoids / Q000096:biosynthesis*; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D013058:Mass Spectrometry / Q000379:methods*; D036226:Seedlings / Q000235:genetics / Q000378:metabolism*
chemical_list: D000872:Anthocyanins; D029681:Arabidopsis Proteins; D005419:Flavonoids
keywords: Anthocyanidin; Arabidopsis thaliana; Deuterated internal standard; Flavonoids; Hydrolysis; LC-MS; Proanthocyanidin; Seed; Seedling