Abstract
Flavonols are a subclass of flavonoids of the group of plant secondary metabolites. In planta, flavonols play various functions such as antioxidant and natural regulator of auxin polar transport. Many lines of evidence have shown that flavonols also contribute to human health in anti-oxidation, anti-inflammation, and even prevention some types of cancer. Several methods have been utilized to measure flavonols such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and diphenylboric acid-2-aminoethyl ester (DPBA) staining. While HPLC or LC-MS can quantitatively determine the level of flavonols, DPBA staining can provide an in-situ view of flavonols accumulation in the plants. In this protocol, a detailed procedure for staining the flavonols in Arabidopsis root tips is described. Five-day-old Arabidopsis seedlings are soaked in a solution containing DPBA and latterly the flavonols (kaempferol and quercetin) can be observed under a confocal microscope.
Keywords: DPBA staining, Kaempferol, Flavonoids, Flavonols, Quercetin
Background
In Arabidopsis, flavonols are biosynthesized from a condensation reaction between one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA. Lewis et al. (2011) found that quercetin, but not kaempferol, is an inhibitor of root basipetal auxin transport. In addition, flavonols also function as an efficient antioxidant for plants and humans as well. A prior study investigated that DPBA can fluoresce when it interacts with flavonols (Sheahan and Rechnitz, 1992). Based on this, DPBA has been widely applied to detect the flavonols accumulation in the plants (Nguyen et al., 2013, 2015 and 2016; Vu et al., 2015). Here, a detailed protocol for DPBA-based detection of flavonols is described. Apart from Arabidopsis, this protocol can be also used for other plants such as Brassica napus L. (Vu et al., 2015). Since other plant species may have a thicker and larger tissue than Arabidopsis, a vacuum can be applied to facilitate the penetration of DPBA into the plant tissues.
Materials and Reagents
Equipment
Procedure
Data analysis
Arrange the data as following order: (1) Bright field; (2) Kaempferol; and (3) Quercetin. Please check some previous publications for details (Nguyen et al., 2013, 2015 and 2016; Vu et al., 2015). Figure 2. Accumulation of flavonols (kaempferol and quercetin) in the root tips of 5-day-old Arabidopsis seedlings (wild-type). Scale bar = 100 μm.
Recipes
Acknowledgments
I would like to appreciate Dr. Cuong Thach Nguyen (Nguyen Tat Thanh University, Vietnam) and Dr. Minh Tan Nguyen (UCLA School of Dentistry, US) for their critical reading this protocol. This protocol was derived from previous publications (Nguyen et al., 2013, 2015 and 2016). The authors declare that they have no conflict of interest.
References
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