Biochemistry

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.

The genus Piper (Piperaceae) is widely distributed in the tropical and subtropical regions of the world, and species belonging to this genus are included in the Ayurvedic system of medicine and in folklore medicine of Latin America. Phytochemical investigations of Piper species have led to the isolation of several classes of physiologically active compounds such as alkaloids, amides, pyrones, dihydrochalcones, flavonoids, phenylpropanoids, lignans and neolignans. In an ongoing investigation of bioactive secondary metabolites from Piper species, herein, we describe the isolation procedure of nine flavonoids, including two chalcones and two flavanones from the leaves of Piper delineatum Trel. (Piperaceae), a shrub native to tropical regions of the Americas. All compounds were elucidated by spectroscopic and spectrometric methods, and comparison with data reported in the literature.

Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important role in bacterial biofilm formation and virulence factor production in many pathogenic strains, QS disruptors have become a hot topic in current antimicrobial research. There are several reporter strains exhibiting QS-regulated phenotypes that have been engineered for the identification of QS inhibitors, including, for example, pigment production (González and Keshavan, 2006; Steindler and Venturi, 2007), gfp, lacZ or lux reporter gene fusions (González and Keshavan, 2006; Steindler and Venturi, 2007), or lethal gene fusions downstream QS-controlled promoters (Weiland-Bräuer et al., 2015). With three parallel QS circuits, the bioluminescent marine bacterium Vibrio campbellii (formerly harveyi, Lin et al., 2010) constitutes a complex Gram-negative model for which an extensive body of knowledge exists, including an array of mutant biosensors. In V. campbellii, bioluminescence is regulated by QS. However, bioluminescence is the result of complex biochemical networks that converge with cell respiration and fatty acid metabolism. It is also an energy-demanding reaction that strongly depends on the overall metabolic state of the bacterium, consuming up to 1/5 of the cell resources (Munn, 2011). Thus, disruption of QS-controlled phenotypes might be the result of toxic side effects or interference with the above-mentioned biochemical pathways rather than QS signaling. Therefore, adequate control experiments should be included. The protocol described herein provides a method and workflow for the identification of putative QS-disrupting compounds in Vibrio. It can also be easily adapted for other QS studies (e.g., detection of AI molecules).

Anthocyanins are a class of flavonoids and important plant pigments. They attract insects to pollinate flowers, protect plants from UV irradiation, and act as antimicrobial agents against herbivores and pathogens. Biosynthesis of anthocyanin is stimulated by diverse developmental signals and environmental stresses including drought, wounding, pathogen infection and insect attack. Plant hormones such as jasmonates, a stress-related plant hormone, also induce accumulation of anthocyanins. Sensitivity of plants to these stress stimuli can be measured by accumulation of anthocyanins. Here we describe a simple method for measurement of anthocyanins in Arabidopsis thaliana seedlings. Amount of anthocyanins are calculated only from absorbances at 530 and 657 nm of crude extract.

This method describes the extraction of coumarins and furanocoumarins from leaves of Ruta graveolens (a natural furanocoumarin) producer, and Nicotiana benthamiana.