Analysis of Phloem Sterols in Plants using EDTA-Facilitated Exudation and GC-MS

Phytosterols occur in the phloem of vascular plants as free, acylated, and glycosylated derivatives. The phloem is increasingly recognized as an active site of sterol synthesis and transport used to support plant metabolism, while also providing a potential nutritional source for phloem-feeding insects. This chapter describes a method for the collection and quantitative analysis of phloem sterols using an EDTA-facilitated exudation procedure followed by gas chromatography-mass spectrometry (GC-MS). Excised leaves are incubated in an EDTA solution to induce phloem exudation, and sterols from collected exudate are extracted, hydrolyzed to their free forms, derivatized as trimethylsilyl ethers, and analyzed by GC-MS using internal standards for quantification. This method has been successfully applied to several plant species and can be adapted to others with suitable modifications. It provides a reproducible approach for characterizing sterol composition of phloem sap and assessing its biological relevance in plant-insect interactions.