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A simple and efficient method for betalain quantification in RUBY-expressing plant samples.
Dibyajyoti Pramanik1,2, Keunsub Lee1,2, Kan Wang1,2
1Department of Agronomy, Iowa State University, Ames, IA, United States.
Frontiers in Plant Science
|October 3, 2024
Summary
A new method quantifies RUBY reporter gene expression in plants by measuring betalain content. This rapid protocol enables accurate comparative analysis of gene expression in transgenic tissues without sample sacrifice.
Area of Science:
- Plant biotechnology
- Molecular biology
- Reporter gene systems
Background:
- The RUBY reporter system offers a visible marker for gene expression in plants, producing red pigment.
- Existing visual markers like GUS and fluorescent proteins often require sample destruction or specialized equipment.
- A lack of robust quantitative methods for betalain content has hindered accurate analysis of RUBY expression.
Purpose of the Study:
- To develop a simple, rapid protocol for quantifying betalain content to evaluate RUBY reporter gene expression.
- To validate the protocol's effectiveness in both transiently and stably transformed plant tissues.
- To establish a reliable method for comparative analysis of gene expression driven by different regulatory elements.
Main Methods:
- Developed a protocol for rapid betalain extraction from fresh plant tissues using a hand grinder and plastic pestle, avoiding liquid nitrogen.
- Quantified betalain content using spectrophotometry and High-Performance Liquid Chromatography (HPLC).
- Correlated betalain content with RUBY expression levels in agroinfiltrated *Nicotiana benthamiana* leaves and transgenic maize tissues (seeds, leaves, roots).
Main Results:
- The developed method provides rapid and consistent betalain quantification, validated by spectrophotometric and HPLC analyses.
- Differential RUBY expression was successfully quantified in *N. benthamiana* leaves (110–1066 mg/kg) and maize tissues (15.3–1028.7 mg/kg).
- Betalain content strongly correlated with RUBY expression levels, indicating suitability for monitoring transformation efficiency.
Conclusions:
- The presented protocol offers a simple, rapid, and accurate method for quantifying RUBY reporter gene expression in various plant tissues.
- This method eliminates the need for sample sacrifice or specialized equipment, streamlining comparative studies.
- The protocol is expected to advance research on plant promoters, enhancers, and other regulatory elements in transgenic systems.
