Reticulophagy, the selective autophagy of endoplasmic reticulum (ER) components, is known to operate in eukaryotes from yeast and unicellular algae to animals and plants. Thus far, only ER-stress induced reticulophagy was reported and analyzed in plants. In this study we characterize a reticulophagy pathway in Arabidopsis thaliana that is triggered by dark-induced starvation but not by ER stress. This pathway is defined by the previously reported ATG8-interacting proteins, ATI1 and ATI2. We further identified the ER-localized MSBP1 (Membrane Steroid Binding Protein 1) as an ATI1- and ATI2-interacting protein and an autophagy cargo, and show that ATI1 and ATI2 serve as its cargo receptors. Together, these findings expand our knowledge on plant responses during energy deprivation and highlight the role of this special type of reticulophagy in this process. Abbreviations: AGO1: ARGONAUTE 1; ATI: ATG8-Interacting Protein; BiFC: Bimolecular Fluorescence Complementation; BR: brassinosteroid; conA: concanamycin A; DMSO: dimethyl sulfoxid; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAPR: Membrane-Associated Progesterone Binding Protein; MSBP: Membrane Steroid Binding Protein; SD: standard deviation; SE: standard error; TM: tunicamycin; TOR: target of rapamycin; Y2H: yeast two-hybrid.

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life of crops have generally been associated with reduced levels of volatile specialized metabolites and hence with decreased aroma and flavor. Thus, there is a need for the development of new varieties that will retain their desired traits while gaining enhanced scent and flavor. Metabolic engineering holds great promise as a tool for improving the profile of emitted volatiles of domesticated crops. This mini review discusses recent attempts to utilize metabolic engineering of the phenylpropanoid and its primary precursor pathway to enhance the aroma and flavor of flowers and fruits.

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.

Diatoms are unicellular algae characterized by silica cell walls. These silica elements are known to be formed intracellularly in membrane-bound silica deposition vesicles and exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements remains unknown. Here we study the membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during its formation, the mineral phase is in tight association with the silica deposition vesicle membranes, which form a precise mold of the delicate geometrical patterns. We find that during exocytosis, the distal silica deposition vesicle membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. We demonstrate that within the cell, the proximal silica deposition vesicle membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.

The proper folding of multispanning membrane proteins (MPs) hinges on the accurate insertion of their transmembrane helices (TMs) into the membrane. Predominantly, TMs are inserted during protein translation, via a conserved mechanism centered around the Sec translocon. Our study reveals that the C-terminal TMs (cTMs) of numerous MPs across various organisms bypass this cotranslational route, necessitating an alternative posttranslational insertion strategy. We demonstrate that evolution has refined the hydrophilicity and length of the C-terminal tails of these proteins to optimize cTM insertion. Alterations in the C-tail sequence disrupt cTM insertion in both E. coli and human, leading to protein defects, loss of function, and genetic diseases. In E. coli, we identify YidC, a member of the widespread Oxa1 family, as the insertase facilitating cTMs insertion, with C-tail mutations disrupting the productive interaction of cTMs with YidC. Thus, MP sequences are fine-tuned for effective collaboration with the cellular biogenesis machinery, ensuring proper membrane protein folding.