Abstract
Background(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.ResultsIn 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.ConclusionsWhen 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0880-7) contains supplementary material, which is available to authorized users.
Highlights
Flavonoids play a role in a wide variety of biological phenomena
Optimizing for high throughput For optimizing this method for high throughput analysis we considered four aspects: 1. minimizing the time for LC-MS runs and data management, 2. covering a maximum of selected substances at quantifiable levels, 3. minimizing experimental error and 4. reducing the amount of plant material
We aimed to reduce this hydrolysis time to minimize the degradation of core substances while completely hydrolysing the most abundant glycosylated flavonoids
Summary
Flavonoids play a role in a wide variety of biological phenomena. In plants, their bright colors attract pollinators while their antioxidant properties offer protection from harmful UV-radiation. Proanthocyanidins from red wine have been discussed to explain the “French paradox” – the co-occurrence of low coronary heart disease deaths and a diet rich in saturated fat [1,2,3]. Flavonoids are phenylpropanoid-derived secondary metabolites that may accumulate in various plant tissues. Their production is often regulated by environmental factors including light, temperature, pathogen attack and nutrient deprivation. The major subgroups comprise chalcones, flavones, flavonols, flavandiols, anthocyanidins and proanthocyanidins [6]
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