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To survive extreme desiccation, seeds enter a period of quiescence that can last millennia. Seed quiescence involves the accumulation of protective storage proteins and lipids through unknown adjustments in protein homeostasis (proteostasis). Here, we show that mutation of all six type–II metacaspase (MCA–II) proteases in Arabidopsis thaliana disturbs proteostasis in seeds. MCA–II mutant seeds fail to restrict the AAA ATPase CELL DIVISION CYCLE 48 (CDC48) at the endoplasmic reticulum to discard misfolded proteins, compromising seed storability. Endoplasmic reticulum (ER) localization of CDC48 relies on the MCA–IIs-dependent cleavage of PUX10 (ubiquitination regulatory X domain–containing 10), the adaptor protein responsible for titrating CDC48 to lipid droplets. PUX10 cleavage enables the shuttling of CDC48 between lipid droplets and the ER, providing an important regulatory mechanism sustaining spatiotemporal proteolysis, lipid droplet dynamics, and protein homeostasis. In turn, the removal of the PUX10 adaptor in MCA–II mutant seeds partially restores proteostasis, CDC48 localization, and lipid droplet dynamics prolonging seed lifespan. Taken together, we uncover a proteolytic module conferring seed longevity. Here, the authors have generated a metacapase type II depletion model providing evidence for their paramount role in seed longevity. They also show that this is accomplished by regulating the ERAD, the proteostatic pathway crucial for seeds. [ABSTRACT FROM AUTHOR]

Tudor staphylococcal nuclease (TSN; also known as Tudor‐SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant‐specific SG‐localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein–protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat‐induced activation of the evolutionarily conserved energy‐sensing SNF1‐related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP‐activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling. SYNOPSIS: Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved component of stress granules (SGs). Here, TSN is found to serve both as a scaffold for previously uncharacterized components of plant stress granules, and as a positive regulator of SnRK1/AMPK signalling in Arabidopsis. TSN is part of an extensive protein‐protein interaction network enriched for intrinsically‐disordered proteins before onset of stress.TSN recruits diverse proteins to SGs via its highly disordered N‐terminal region comprising four SN domains.TSN interacts with the catalytic α‐subunit of SnRK1 and enhances its kinase activity during heat stress.Proteomics data identify plant‐specific SG components. [ABSTRACT FROM AUTHOR]

Membrane trafficking is central to cell plate construction during plant cytokinesis. Studies on cell plate formation can provide answers to basic biology questions including molecular mechanisms of membrane trafficking, tissue patterning, and cytoskeletal dynamics. Consequently, a detailed understanding of cytokinesis depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles and delivery of proteins to the developing and maturing plate. This chapter describes a pipeline based on fluorescence recovery after photobleaching (FRAP) to measure and analyze turnover of peripheral or transmembrane proteins on the cell plate. The approach described here can also be applied in other biological contexts., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)