Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.myosin XI | actin filament | cytoplasmic streaming | velocity distribution map | Arabidopsis thaliana C ytoplasmic streaming, defined as an extensive intracellular motility in plants, was first described in 1774 (1). It is thought that unidirectional actin filament (AF) bundles and organelleassociated myosin XI, a plant-specific class of myosin motors, cause bulk flow in the cell (reviewed in refs. 2-4). Some myosin XI were indeed reported to slide along AFs in vitro (5, 6). Using immunocytochemical analyses (7-11) and fluorescent proteinlabeled myosins (12, 13), myosin XI have been shown to be associated with the particulate organelles. Recent analyses using gene knockouts and dominant-negative inhibition demonstrated that several class XI myosins have overlapping functions in the rapid movement of organelles (14-18). Among these, myosin XI-K was found to play the most prominent role in the movement of Golgi bodies, peroxisomes, and mitochondria. Studies have also concluded that none of these three organelles fits the paradigm of cytoplasmic streaming, raising the question of what drives this conspicuous process (14, 15).The endoplasmic reticulum (ER), an organelle present in all eukaryotic cells, harbors the largest reservoir of cellular membranes. Cytoskeleton-dependent remodeling of the ER network, which consists of cisternae and interconnected membrane tubes, is well known in both animals and plants (reviewed in refs. 19-22). In addition, dynamic streaming of the plant ER has been observed in subperipheral cytoplasm and transvacuolar strands (23-25). However, the molecular mechanism underlying the ER streaming is not known, although it was reported that the 175 kDa myosin XI was localized on the ER of tobacco cell culture BY-2 (26).Unlike the movement of the part...

The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a crucial process for various cellular activities.The NPC comprises ;30 nucleoporins and is well characterized in vertebrates and yeast. However, only eight plant nucleoporins have been identified, and little information is available about the complete molecular structure of plant NPCs. In this study, an interactive proteomic approach was used to identify Arabidopsis thaliana nucleoporins. A series of five cycles of interactive proteomic analysis was performed using green fluorescent protein (GFP)-tagged nucleoporins. The identified nucleoporins were then cloned and subcellular localization analyses were performed. We found that the plant NPC contains at least 30 nucleoporins, 22 of which had not been previously annotated. Surprisingly, plant nucleoporins shared a similar domain organization to their vertebrate (human) and yeast (Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins exhibited higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358], Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis failed to identify a vertebrate homolog for this protein. Interestingly, Nup136-GFP showed greater mobility on the nuclear envelope than did other nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant development. These findings provide valuable new information about plant NPC structure and function.

The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1͞AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca 2؉ -dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.

Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cellautonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.[Keywords: Membrane fusion; proteasome; cell-autonomous immunity; hypersensitive response; caspase activity; programmed cell death] Supplemental material is available at http://www.genesdev.org.