Visualization and Quantification of Metabolite Dynamics in Salt‐Stressed Wheat Leaves via Dual‐Excitation Raman Spectral Imaging

ABSTRACT

Wheat is one of the most important staple crops worldwide, and its yield and quality are severely compromised by soil salinization. Metabolic homeostasis in leaves plays a pivotal role in plant growth and stress adaptation; however, the spatiotemporal metabolic dynamics underlying salt‐induced leaf senescence remain largely elusive. In this study, Raman spectral imaging equipped with 532‐ and 785‐nm excitation lasers was employed to characterize metabolite fluctuations and their spatial distribution patterns in salt‐stressed wheat leaves. Salt stress markedly decreased the abundance of carotenoids, chlorophyll, lipids, carbohydrates, and phenylpropanoids, and sequential degradation profiles of these metabolites were revealed by Pearson correlation analysis. The 532‐nm excitation induced resonance Raman enhancement, allowing high‐contrast visualization of metabolite localization in leaf tissues, showing tissue‐specific accumulation of pectin at leaf margins and phenylpropanoids in vascular bundles. Principal component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA) efficiently discriminated different senescence stages, with carotenoids identified as key diagnostic biomarkers. Using lutein as an external reference, we constructed quantitative calibration curves. The average relative error was 8.61% for Raman spectroscopy and 2.095% for Raman imaging, enabling visualization and relative quantitative analysis of the target metabolite. This study validates the capability of Raman spectral imaging for real‐time, spatially resolved, and quantitative profiling of plant metabolites, offering a promising approach for assessing plant physiological status under saline stress.