No abstract

Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.

Despite recent progress in our understanding of graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy (CLEM) approach to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed efficient targeting of the grafting interface for examination under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the cell wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these incomplete PD gives us insights into the process of secondary PD biogenesis. We found that the establishment of successful symplastic connections between the scion and rootstock occurred predominantly in the presence of thin cell walls and endoplasmic reticulum–plasma membrane tethering. The resolution reached in this work shows that our CLEM method advances the study of biological processes requiring the combination of light and electron microscopy.

Endocytosis is the process by which cells internalise molecules from their cell surface via plasma membrane-derived vesicles. In plants, clathrin-mediated endocytosis requires the evolutionarily ancient TSET/TPLATE complex (TPC), which was lost in metazoan and fungal lineages. TPC is required for membrane bending, but how TPC functions in the initiation of endocytosis and clathrin assembly is unclear. Here we used live-cell imaging and biochemical approaches to investigate the function of the Arabidopsis thaliana TPC subunit AtEH1/Pan1. Using in vitro and in vivo experiments we found that AtEH/Pan1 proteins can self-assemble into condensates through phase separation, which is influenced by both structured and intrinsically disordered regions. The proteome composition of these condensates revealed many key endocytic components which are selectively recruited via prion-like- and IDR-based interactions, including the ESCRT-0 TOM-Like proteins. Furthermore, AtEH/Pan1 condensates selectively nucleate on the plasma membrane by binding specific phospholipid species that are recognised by their EH domains. Visualization of the ultrastructure of the endocytic condensates via CLEM-ET revealed that the coat protein clathrin can assemble into lattices within condensates. Our results reveal that AtEH/Pan1 proteins act as scaffolds to direct endocytic machinery to specific plasma membrane regions to initiate internalisation. These findings provide new insight into the interplay between membranes and protein condensates.

Throughout their life cycle, plants face a tremendous number of environmental and developmental stresses. To respond to these different constraints, they have developed a set of refined intracellular systems including autophagy. This pathway, highly conserved among eukaryotes, is induced by a wide range of biotic and abiotic stresses upon which it mediates the degradation and recycling of cytoplasmic material. Central to autophagy is the formation of highly specialized double membrane vesicles called autophagosomes which select, engulf, and traffic cargo to the lytic vacuole for degradation. The biogenesis of these structures requires a series of membrane remodeling events during which both the quantity and quality of lipids are critical to sustain autophagy activity. This review highlights our knowledge, and raises current questions, regarding the mechanism of autophagy, and its induction and regulation upon environmental stresses with a particular focus on the fundamental contribution of lipids. How autophagy regulates metabolism and the recycling of resources, including lipids, to promote plant acclimation and resistance to stresses is further discussed.

No abstract

In plants, macroautophagy/autophagy is a key mechanism that contributes to their ability to cope with a wide range of environmental constraints such as drought, nutrient starvation or pathogen resistance. Nevertheless, the molecular mechanisms of plant autophagy, and notably that of autophagosome formation, remain poorly understood. As the starting point of our recent paper, we considered the potential functional contribution of lipids in the numerous membrane-remodeling steps involved in this process. By combining biochemistry, genetics, cell biology and high-resolution 3D imaging, we unraveled the function of the lipid phosphatidylinositol-4-phosphate (PtdIns4P) in autophagy in Arabidopsis thaliana, thus providing novel insights into the assembly of autophagosomes in plant cells.

Despite recent progress in our understanding of the graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy approach (CLEM) to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed the efficient targeting of the grafting interface for under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the call wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these failed PD gives us insights into the process of secondary PD biogenesis. We showed that the thinning of the cell wall and the endoplasmic reticulum-plasma membrane tethering seem to be required for the establishment of symplastic connections between the scion and the rootstock. The resolution reached in this work shows that our CLEM method offer a new scale to the study for biological processes requiring the combination of light and electron microscopy.