Analysis of Citrate Distribution in Plant Tissues Using Multimodal Imaging

Significance of the topic

The spatial mapping of small organic acids such as citrate in plant tissues is essential for understanding fruit flavor, metabolic zonation, transport, and nutrient allocation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI‑MSI) is widely used to visualize metabolite distributions, but ions are commonly observed as metal-adducts (e.g., [M+K]+), which raises concerns that heterogeneous inorganic ion distributions could distort the apparent metabolite maps. This study applies a multimodal approach combining MALDI‑MSI, laser ablation‑inductively coupled plasma‑MS imaging (LA‑ICP‑MS), and targeted HPLC quantification to assess whether the MALDI positive‑ion image of citrate in strawberry sections reflects true citrate concentration or is biased by potassium heterogeneity.

Objectives and overview of the study

The main goals were to (1) image citrate distribution in strawberry fruit tissue by MALDI‑MSI in positive‑ion mode where citrate appears as a potassium adduct, (2) image potassium distribution on the same tissue by LA‑ICP‑MS, and (3) quantitatively determine citrate content in adjacent tissue pieces using a dedicated organic acid HPLC system (Nexera Organic Acid Analysis System). By correlating MALDI signal intensity with HPLC concentrations and comparing spatial potassium maps, the study tests whether MALDI [M+K]+ images are artifacts of potassium heterogeneity.

Used Instrumentation

• MALDI imaging: iMScope QT coupled with LCMS‑9050; matrix deposition with iMLayer AERO (2,5‑DHB in 50% MeOH + 0.2% TFA).
• LA‑ICP imaging: imageBIO266 laser ablation coupled to ICPMS‑2050 for elemental mapping (potassium, m/z 39).
• Quantitative organic acid analysis: Nexera Organic Acid Analysis System with Shim‑pack SCR‑102H guard and analytical columns; conductivity detection.
• Data integration and visualization: IMAGEREVEAL MS software for both MS imaging modalities.

Methodology

• Sample preparation: Fragaria × ananassa ‘Tochiotome’ fruits were cryosectioned (90 µm) and mounted on ITO slides. DHB matrix was spray‑coated (four layers) prior to MALDI‑MSI in positive polarity (m/z 190–250), spatial pitch 50 µm.
• MALDI‑MSI acquisition targeted citrate-related signals appearing as [M+K]+; instrument parameters were optimized for tissue imaging.
• After MALDI imaging, the identical section was subjected to LA‑ICP‑MS to map elemental potassium distribution, using laser parameters yielding ~55 µm ablation spot size and an ICP RF power of ~1.2 kW.
• For quantitative validation, twelve adjacent tissue pieces (2 × 4 × 0.56 mm) were excised from the imaged region, freeze‑dried, homogenized, extracted in water with heating (70 °C, 30 min), centrifuged, diluted 5× and analyzed by the Nexera organic acid system using external aqueous citrate standards (2.5–50 mg/L).
• Image and data analysis were performed in IMAGEREVEAL MS; average ion intensities per region were computed and regressed against HPLC citrate concentrations.

Main results and discussion

• MALDI‑MSI: Citrate appeared primarily as its potassium adduct and showed higher signal intensity in the outer cortex (calyx side) and lower intensity toward the pith (central region), with the strongest signals in the most peripheral tissue pieces.
• LA‑ICP‑MS (potassium): Potassium abundance was elevated in the outer cortex and vascular bundles, but its detailed spatial pattern did not spatially match the citrate [M+K]+ MALDI image on the same section.
• Quantitative HPLC: Citrate concentrations measured in the 12 excised tissue pieces showed the highest citrate content in the outermost cortex and decreasing levels toward the inner tissue—replicating the MALDI image trend.
• Correlation analysis: A strong linear correlation (R2 > 0.96) was observed between region‑averaged MALDI [M+K]+ ion intensities and HPLC‑quantified citrate concentrations. This demonstrates that the MALDI positive‑ion image faithfully represents citrate distribution despite heterogeneous potassium distribution.

Benefits and practical applications of the method

• Multimodal imaging (MALDI‑MSI + LA‑ICP‑MS) allows simultaneous assessment of metabolite localization and elemental context on the same tissue, enabling validation of adduct‑based signals.
• High concordance with targeted HPLC quantification provides confidence that positive‑ion MALDI images of citrate can be used quantitatively for spatial metabolomics in plant tissues under similar conditions.
• Practical applications include studies of fruit quality and flavor, spatial metabolism in developmental biology, plant nutrient research, breeding selection, and food science/QA where localized metabolite levels are important.

Limitations and considerations

• This work focused on a single plant species and matrix/ionization conditions; results may differ with other tissues, matrices, or ion suppression scenarios.
• The study used external HPLC calibration on adjacent tissue pieces rather than absolute on‑section MSI quantification; matrix effects and spot‑level variability still warrant careful evaluation for absolute quantitation by MSI.
• Potassium heterogeneity observed did not distort citrate imaging in this case, but other analyte–adduct combinations or stronger elemental gradients could produce artifacts; independent elemental imaging remains valuable for validation.

Future trends and potential applications

• Advances in on‑section quantification methods, isotope‑labeled internal standards, and calibration strategies will improve absolute MSI quantification for metabolites.
• Higher spatial resolution MALDI and LA‑ICP instruments will enable subcellular and single‑cell correlative maps of metabolites and elements.
• Integrated multimodal workflows combining MSI, elemental imaging, optical microscopy and spatial transcriptomics will deepen mechanistic understanding of plant physiology and metabolite transport.
• Automated data fusion, machine learning and standardized pipelines for cross‑platform registration (e.g., IMAGEREVEAL MS enhancements) will increase throughput and reproducibility for applied research and industry use.

Conclusion

Multimodal imaging combining positive‑ion MALDI‑MSI, LA‑ICP‑MS and targeted HPLC quantification demonstrates that the MALDI [M+K]+ signal for citrate in strawberry fruit reliably reflects the underlying citrate concentration distribution, rather than being an artifact of heterogeneous potassium. The integrated approach validates adduct‑based MSI readouts and supports the wider use of MALDI‑MSI for spatial metabolite studies in plant tissues when coupled with complementary elemental and quantitative analyses.

References

• Enomoto et al., Journal of Agricultural and Food Chemistry, 66: 4958–4965 (2018).