Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Posttranscriptional gene silencing (PTGS) mediated by siRNAs is an evolutionarily conserved antiviral defense mechanism in higher plants and invertebrates. In this mechanism, viral-derived siRNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. In Arabidopsis, a key component of RISC is ARGONAUTE1 (AGO1), which not only binds to siRNAs but also carries the RNA slicer activity. At present little is known about posttranslational mechanisms regulating AGO1 turnover. Here we report that the viral suppressor of RNA silencing protein P0 triggers AGO1 degradation by the autophagy pathway. Using a P0-inducible transgenic line, we observed that AGO1 degradation is blocked by inhibition of autophagy. The engineering of a functional AGO1 fluorescent reporter protein further indicated that AGO1 colocalizes with autophagy-related (ATG) protein 8a (ATG8a) positive bodies when degradation is impaired. Moreover, this pathway also degrades AGO1 in a nonviral context, especially when the production of miRNAs is impaired. Our results demonstrate that a selective process such as ubiquitylation can lead to the degradation of a key regulatory protein such as AGO1 by a degradation process generally believed to be unspecific. We anticipate that this mechanism will not only lead to degradation of AGO1 but also of its associated proteins and eventually small RNAs.

The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H + -pyrophosphatase and the vacuolar H + -adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.