Spectrophotometric Quantification of Betacyanins in Plant Tissue Produced from the RUBY Reporter Gene
Matthew Morgan
Abstract
RUBY is a visual reporter gene that produces beatnin, a betacyanin that appears red to violet in colour (He et al., 2020). To assay betanin (and other betalains) a spectrophotometric approach has been employed, as seen in Stintzing et al. (2003) and Grützner et al. (2021).
Steps
Sample collection
Punch out leaf discs (as many as possible in the target area) of areas expressing betanin.
Put leaf discs in 2 ml micro-centrifuge tubes and snap freeze in liquid nitrogen then store in -80 freezer until required.
Sample Preparation
Take 20-50 mg of frozen tissue and add to 2 ml tubes. Keep on ice.
Grind down tissue using plastic micro-pestle and add methanol buffer (50% methanol, 1 mM ascorbic acid, 0.5% formic acid) at 10% w/v - i.e. for 35 mg of tissue add 350 ul methanol buffer.
Dilute samples with mili-Q so that absorption values fall within 0.8 - 1.0 (as previously calculated by Stintzing et al. 2003). Grützner et al. (2021) found that a 12 fold dilution performed well but this could differ.
Spectrophotometric Quantifications
Put cuvettes or 96 well plates into spectophotometer and read absorbtion at 538 nm (Stintzing et al. 2003).
Using the formula BC = (A * DF * MW * 1000) / ( ε x L) calculate the concentration of betacyanins.
BC = betanin content (mg/L) A = absorbance (538 nm) DF = dilution factor MW = molecular weight (550.47 g/mol) L = cuvette path length ε = molar extinction coefficient (60, 000 L/(mool cm)
