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<p>Extracellular vesicles (EVs) are crucial mediators of intercellular communication in various biological systems, including plants, where they facilitate the sharing of proteins, lipids, and nucleic acids. While plant-derived EVs (PDEVs) have documented intrinsic functions in stress and host–pathogen responses in model species, methods for isolating and characterizing these vesicles from agriculturally important crops, such as cucumber (<i>Cucumis sativus</i>) apoplast, remain unoptimized. This methodological study evaluates and compares current protocols to establish an efficient and standardized technique for PDEV isolation from apoplastic washing fluid (AWF). We directly compared three key isolation methods PEG precipitation, ultracentrifugation, and size exclusion chromatography (SEC) assessing their performance based on cost, scalability, purity, and vesicle preservation/integrity. Transmission electron microscopy (TEM) and immunoblot analyses revealed the morphology and proteome profile of the isolated EVs, validating vesicle integrity and marker enrichment obtained from each method. Results confirm that while ultracentrifugation serves as the gold standard for isolation, it risks compromising vesicle integrity. Conversely, PEG precipitation offers a low-cost, scalable method, despite co-precipitating soluble proteins. We demonstrate the value of using SEC as a critical secondary purification step to enhance the purity of the isolated EVs. In conclusion, TEM and immunoblot characterization together supported that SEC-based purification yields the most morphologically intact and biochemically enriched EVs. We further demonstrate the cross-species applicability of AtTET8 and AtPEN1 antibodies in cucumber and validate CsTET8 as an EV-associated marker, underscoring the importance of standardized workflows for crop PDEV research. Establishing a reliable, optimized protocol is an essential first step to enable rigorous functional studies of crop EVs and to unlock their potential applications in both biomedicine and sustainable agriculture.</p>

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Optimization of isolation and characterization methods for extracellular vesicles from Cucumis sativus apoplast

  • 期刊论文

  • 发表日期：

    2026年5月21日

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  • Neha Devi,
  • Nisha Devi,
  • Ravi Gupta,
  • Sunny Dhir,
  • Yashika Walia

摘要

Extracellular vesicles (EVs) are crucial mediators of intercellular communication in various biological systems, including plants, where they facilitate the sharing of proteins, lipids, and nucleic acids. While plant-derived EVs (PDEVs) have documented intrinsic functions in stress and host–pathogen responses in model species, methods for isolating and characterizing these vesicles from agriculturally important crops, such as cucumber (Cucumis sativus) apoplast, remain unoptimized. This methodological study evaluates and compares current protocols to establish an efficient and standardized technique for PDEV isolation from apoplastic washing fluid (AWF). We directly compared three key isolation methods PEG precipitation, ultracentrifugation, and size exclusion chromatography (SEC) assessing their performance based on cost, scalability, purity, and vesicle preservation/integrity. Transmission electron microscopy (TEM) and immunoblot analyses revealed the morphology and proteome profile of the isolated EVs, validating vesicle integrity and marker enrichment obtained from each method. Results confirm that while ultracentrifugation serves as the gold standard for isolation, it risks compromising vesicle integrity. Conversely, PEG precipitation offers a low-cost, scalable method, despite co-precipitating soluble proteins. We demonstrate the value of using SEC as a critical secondary purification step to enhance the purity of the isolated EVs. In conclusion, TEM and immunoblot characterization together supported that SEC-based purification yields the most morphologically intact and biochemically enriched EVs. We further demonstrate the cross-species applicability of AtTET8 and AtPEN1 antibodies in cucumber and validate CsTET8 as an EV-associated marker, underscoring the importance of standardized workflows for crop PDEV research. Establishing a reliable, optimized protocol is an essential first step to enable rigorous functional studies of crop EVs and to unlock their potential applications in both biomedicine and sustainable agriculture.