Isolation and Visualization of Plant Stress Granule-Associated Components via On-Beads Digestion and Co-localization Analysis.

Stress granules (SGs) are conserved cytoplasmic biomolecular condensates mainly formed by proteins and RNA molecules assembled by liquid-liquid phase separation. Isolation of SGs components has been a major challenge in the field due to the dynamic and transient nature of stress granule shells. Here, we describe the methodology for the isolation and visualization of SGs proteins from Arabidopsis thaliana plants using a scaffold component as the target. The protocol consists of the first immunoprecipitation of GFP-tagged scaffold protein, followed by an on-beads enzymatic digestion and previous mass spectrometry identification. Finally, the localization of selected SGs candidates is visualized in Nicotiana benthamiana mesophyll protoplasts.

Seed longevity is controlled by metacaspases.

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.