Background: The plant endomembrane system comprises the nucleus membrane, plasma membrane and a series of functionally distinct membrane-bounded organelles, including endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), prevacuolar compartment/multivesicular body (PVC/MVB), autophagosome, and vacuole. Through this network, signals and substances, usually proteins are transported within or exchanged between cells, which is essential for the growth and development of plants, and their responses to the environment. In plant cells, protein trafficking pathways can be divided into at least three groups, including the protein secretory pathway, vacuolar trafficking pathway and endocytic pathway. Vesicles are used as carriers and are responsible for accurate cargo sorting, targeting and transport both intra- and inter-cellularly. However, due to the limited resolution of the conventional light microscopes, some of the organelles and vesicles have not yet been clearly observed or even visualized. Debates and gaps persist on the knowledge of important scientific questions of plant endomembrane trafficking. Hence, the application of electron microscopy (EM)-related technologies with nanometer resolution offers great favor to plant scientists.
Progress: Recently, an increasing number of novel and non-classic transport pathways have been identified and demonstrated. Firstly, in addition to the well-known conventional protein secretory pathway (CPS), the unconventional protein secretion (UPS) has also been gradually discovered in plant cells for secretory proteins without signal peptide. The UPS can potentially mediate the formation of plant extracellular vesicles. Secondly, among the vacuolar trafficking, a new pathway which is not by way of Golgi apparatus but directly originates from the ER has been found. As the special type of vacuolar trafficking, autophagy has multiple sources of membranes, and structural evidence from electron tomography (ET) shows that the ER is one of the membrane origins. Thirdly, although cell walls are around plant cells, endocytosis does occur and play physiological functions in plants. Compared with the well-investigated clathrin-mediated endocytosis (CME), emerging results on clathrin-independent endocytosis (CIE) indicate its existence in plant cells, especially when plants receive external stimuli. Aforementioned progress in plant endomembrane trafficking cannot be made without the development and application of EM-related technologies. The cryogenic electron microscopy (cryo-EM) helps decode the 3D structure of ATG9, the only transmembrane ATG protein in Arabidopsis, providing testable assumptions for plant autophagy mechanism study. When the objects of interest are organelles that are more complex and larger than proteins, researchers then take advantage of ET. Based on the reconstructed 3D models, one can unveil the biogenesis of plant organelles. In addition, cryo-focused ion beam (cryo-FIB) is used for thinning large samples for cryo-EM/ET observation, which would have great potential in plant study. Last but not least, the correlative light and electron microscopy (CLEM) optimizes the positioning of target of interest under EM with the help of fluorescent labelling under a light microscope.
Perspective: Despite the growing characterizations of new molecules, new pathways and even unknown organelles, our understanding on the regulatory mechanisms of plant endomembrane trafficking is still in its infancy, and there are a lot of important questions waiting for us to answer. For example, is there crosstalk between different protein trafficking pathways in plant cells? If so, what proteins mediate these interactions? What is the biological significance of these interactions? When plants are facing biotic or abiotic stresses, how would different trafficking pathways coordinate and cooperate so that plants can resist and finally survive? Can we use genetic engineering approach to increase the grain yield and improve crop quality by optimizing specific proteins within the cellular trafficking pathways? To answer these questions, multidisciplinary fields (e.g., mathematics, physics, computer science, chemistry and biology) and new technology development such as cutting-edge EM imaging methods are indispensable. With the optimization of experimental conditions and automated processing of a large amount of data, it is expected to achieve in situ analysis of physiological processes such as vesicle trafficking and organelle biogenesis in plant cells, and to clarify underlying regulatory mechanisms at molecular and even atomic levels in the near future. The advances on plant endomembrane trafficking and the related electron microscopy application in plant cells not only deepen our understanding in this field, but also provide clues for the endomembrane system in animal cells, fungi and other systems, which may eventually be transformed into agriculture and medical therapy, together promoting our sustainable development in future.