Área de Parasitología, Dept. Farmacia y Tecnología Farmacéutica y Parasitología, F. Farmàcia, Universitat de València, Burjassot, Valencia, Spain.
Joint Unit on Endocrinology, Nutrition and Clinical Dietetics, IIS La Fe-Universitat de València, Valencia, Spain.
J Extracell Vesicles. 2024 Oct;13(10):e12517. doi: 10.1002/jev2.12517.
The morphogenesis of higher plants requires communication among distant organs throughout vascular tissues (xylem and phloem). Numerous investigations have demonstrated that phloem also act as a distribution route for signalling molecules being observed that different macromolecules translocated by the sap, including nucleic acids and proteins, change under stress situations. The participation of extracellular vesicles (EVs) in this communication has been suggested, although little is known about their role. In fact, in the last decade, the presence of EVs in plants has originated a great controversy, where major concerns arose from their origin, isolation methods, and even the appropriate nomenclature for plant nanovesicles. Phloem sap exudates from melon plants, either aphid-free or infested with Aphis gossypii, were collected by stem incision. After sap concentration (Amicon), phloem EVs (PhlEVs) were isolated by size exclusion chromatography. PhlEVs were characterised using Nanoparticle Tracking Analysis, Transmission electron microscopy and proteomic analysis. Here we confirm the presence of EVs in phloem sap in vivo and the detection of changes in the particles/protein ratio and composition of PhlEVs in response to insect feeding, revealing the presence of typical defence proteins in their cargo as well as components of the proteasome complex. PhlEVs from infested plants showed lower particles/protein ratio and almost two times more proteolytic activity than PhlEVs from aphid-free plants. In both cases, such activity was inhibited in a dose-dependent manner by the proteasome inhibitor MG132. Our results suggest that plants may use this mechanism to prepare themselves to receive infectious agents and open up the possibility of an evolutionary conserved mechanism of defence against pathogens/stresses in eukaryotic organisms.
高等植物的形态发生需要在维管组织（木质部和韧皮部）中进行远距离器官之间的通讯。大量研究表明，韧皮部也充当信号分子的分配途径，观察到不同的通过汁液易位的大分子，包括核酸和蛋白质，在胁迫情况下会发生变化。已经提出了细胞外囊泡（EVs）在这种通讯中的参与，尽管对其作用知之甚少。事实上，在过去的十年中，植物中外泌体的存在引起了很大的争议，主要关注的问题是它们的起源、分离方法，甚至是植物纳米囊泡的适当命名法。通过茎切口收集无蚜虫或受棉蚜（Aphis gossypii）侵袭的甜瓜植物的韧皮部汁液渗出物。在汁液浓缩（Amicon）后，通过尺寸排阻色谱法分离韧皮部 EVs（PhlEVs）。使用纳米颗粒跟踪分析、透射电子显微镜和蛋白质组学分析对 PhlEVs 进行了表征。在这里，我们在体内证实了 EVs 在韧皮部汁液中的存在，并检测到昆虫取食后 PhlEVs 中颗粒/蛋白质比值和组成的变化，揭示了其货物中存在典型的防御蛋白以及蛋白酶体复合物的成分。与无蚜虫植物的 PhlEVs 相比，受侵染植物的 PhlEVs 的颗粒/蛋白质比值更低，且几乎两倍的蛋白水解活性。在这两种情况下，这种活性都被蛋白酶体抑制剂 MG132 以剂量依赖性方式抑制。我们的结果表明，植物可能利用这种机制来准备接收传染性物质，并为真核生物中针对病原体/胁迫的防御机制的进化保守性开辟了可能性。
