A high-throughput method for the quantitative analysis of auxins.

Area of Science:

• Plant Biology
• Biochemistry
• Analytical Chemistry

Background:

• Accurate quantification of plant hormones is essential for understanding plant growth and development.
• Indole-3-acetic acid (IAA) is the most abundant natural auxin, playing critical roles in various plant processes.
• Existing methods for IAA measurement can be time-consuming or require large sample amounts.

Purpose of the Study:

• To develop and validate a precise, high-throughput protocol for determining free IAA concentrations in plant tissues.
• To utilize isotope dilution analysis coupled with gas chromatography-mass spectrometry (GC-MS) for accurate quantification.
• To establish a method suitable for small sample sizes and large-scale sample processing.

Main Methods:

• Plant material harvesting and preparation.
• Solid-phase purification (amino and polymethylmethacrylate) followed by derivatization with diazomethane (manual or robotic).
• Isotope dilution analysis using [¹³C₆]IAA as the internal standard and GC-MS detection.

Main Results:

• The protocol allows for precise determination of free IAA in small plant tissue samples (25 mg fresh weight).
• High-throughput analysis is achievable, with over 500 samples processed per week using automation.
• Robotic extraction significantly improves time efficiency for processing more than eight samples per batch.

Conclusions:

• This optimized protocol provides a reliable and efficient method for quantifying IAA in plants.
• The high-throughput capability supports large-scale studies on plant hormone signaling and metabolic homeostasis.
• The method is suitable for diverse research applications requiring accurate auxin measurements.