Visualization of Abscisic Acid Distribution in Brown Rice Using Mass Spectrometry Imaging

Importance of the topic

Plant hormones regulate adaptive responses to environmental stresses and key developmental processes at trace levels. Visualizing their spatial distribution within tissues is essential to understand hormone function and regulation. Mass spectrometry imaging (MSI) offers label-free in situ mapping of biomolecules, but detecting low-abundance hormones such as abscisic acid (ABA) requires enhanced ionization efficiency. Chemical derivatization combined with MSI can overcome this challenge, enabling detailed studies of hormone localization in crops.

Aims and overview

This study aims to demonstrate a high-sensitivity MSI approach for visualizing ABA distribution in brown rice seeds. By derivatizing ABA with Girard’s reagent T and imaging with the iMScope QT system, the work seeks to improve detection limits, validate spatial localization in embryo versus endosperm, and compare performance to other derivatization methods and underivatized analysis.

Methodology and instrumentation

• Sample preparation: Brown rice (Oryza sativa cv. Hinohikari) hulls removed, embedded in 10% porcine gelatin, and frozen at −80 °C. Sections (15 µm) cut on a Leica CM1950 cryostat, mounted on ITO-coated slides with conductive tape.
• Derivatization and matrix application: ABA reacted with Girard’s reagent T (5 mg/mL, MeOH/H₂O/TFA 80/20/2) for 30 min at room temperature. The GirT-ABA mixture mixed with DHB matrix (30 mg/mL, 70% MeOH) and sprayed onto tissue using an airbrush. Tissue incubated 30 min, dried under reduced pressure, and final DHB layer applied.
• MS imaging: Data acquired on Shimadzu iMScope QT in positive ion mode (m/z 350–400), pixel size 25 µm. Calibration with DHB standard, MS/MS performed at m/z 378.24 (GirT-ABA precursor) using argon collision. Images processed with IMAGEREVEAL MS software.

Used instrumentation

• iMScope QT imaging mass microscope (Shimadzu)
• IMAGEREVEAL MS data analysis software (Shimadzu)
• Leica CM1950 cryostat
• Cryofilm type2C (SECTION-LAB)
• ITO-coated slides and conductive tape (MATSUNAMI, 3M)
• Airbrush system for matrix application

Main results and discussion

GirT derivatization produced a strong [M]+ ion for ABA at m/z 378.24, delivering significantly higher signal intensity than Girard’s reagent P and non-derivatized ABA in negative mode. MS/MS fragmentation yielded characteristic ions at m/z 319.17 and 291.17, confirming ABA identity. MSI of rice seed sections revealed pronounced ABA accumulation in the embryo compared to the endosperm, with statistically significant differences (p<0.0005). These results establish the derivatization-MSI protocol as a sensitive tool for spatial hormone profiling.

Benefits and practical applications

The derivatization–MSI workflow enables direct, high-resolution mapping of trace plant hormones without labels. It supports studies in plant physiology, stress response, and seed development. In agriculture, the method can advance breeding programs, quality control, and targeted interventions by revealing hormone dynamics in situ.

Future trends and possibilities

• Extension of derivatization strategies to other phytohormones (auxins, gibberellins, cytokinins) for multiplexed imaging.
• Quantitative MSI approaches for absolute hormone concentration mapping.
• Development of ambient MSI techniques for rapid, real-time tissue analysis.
• Integration with metabolomics and phenotyping platforms for system-level insights.

Conclusion

Combining Girard’s reagent T derivatization with iMScope QT imaging provides a robust, high-sensitivity method for visualizing ABA distribution in brown rice seeds. The protocol successfully distinguished hormone localization between embryo and endosperm, demonstrating its potential for comprehensive spatial studies of phytohormones in plant science.

Reference

1) Enomoto H, Sensu T, Yumoto E, Yokota T, Yamane H. Derivatization for detection of abscisic acid and 12-oxo-phytodienoic acid using matrix-assisted laser desorption/ionization imaging mass spectrometry. Rapid Commun Mass Spectrom 32, 1565 (2018)