Apr 20, 2023
Version 1
Spectrophotometric Quantification of Betacyanins in Plant Tissue Produced from the RUBY Reporter Gene V.1
- 1Adelaide University
Protocol Citation: matthew.morgan 2023. Spectrophotometric Quantification of Betacyanins in Plant Tissue Produced from the RUBY Reporter Gene. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j4xrlo5/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 12, 2023
Last Modified: April 20, 2023
Protocol Integer ID: 80365
Keywords: spectrophotometric quantification of betacyanin, ruby reporter gene ruby, betacyanin, betanin, visual reporter gene, other betalain, red to violet, plant tissue, beatnin, violet, spectrophotometric approach, spectrophotometric quantification
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).
Materials
- 2 ml micro-centrifuge tubes
- Mini pestle
- Methanol
- Ascorbic acid
- Formic acid
- Cuvettes/96 well plates
- Spectrophotometer
Safety warnings
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)
Protocol references
Grützner, R., Schubert, R., Horn, C., Yang, C., Vogt, T., Marillonnet, S., 2021. Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits. Frontiers in Plant Science, 12. https://doi.org/10.3389/fpls.2021.682443
Stintzing, F.C., Schieber, A. & Carle, R., 2003. Evaluation of colour properties and chemical quality parameters of cactus juices. European Food Research and Technology, 216. https://doi.org/10.1007/s00217-002-0657-0.
He, Y., Zhang, T., Sun, H., Zhan, H. and Zhao, Y. (2020). A reporter for noninvasively monitoring gene expression and plant transformation.Horticulture Research, 7. doi:https://doi.org/10.1038/s41438-020-00390-1.