A rich and bountiful harvest: Key discoveries in plant cell biology.

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Thylakoid-Bound Polysomes and a Dynamin-Related Protein, FZL, Mediate Critical Stages of the Linear Chloroplast Biogenesis Program in Greening Arabidopsis Cotyledons.

Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression analyses of chloroplast proteins. Our study identified a linear developmental sequence with five assembly stages: tubulo-vesicular prothylakoids (24 h after imbibition [HAI]), sheet-like pregranal thylakoids that develop from the prothylakoids (36 HAI), proliferation of pro-grana stacks with wide tubular connections to the originating pregrana thylakoids (60 HAI), structural differentiation of pro-grana stacks and expanded stroma thylakoids (84 HAI), and conversion of the pro-grana stacks into mature grana stacks (120 HAI). Development of the planar pregranal thylakoids and the pro-grana membrane stacks coincides with the appearance of thylakoid-bound polysomes and photosystem II complex subunits at 36 HAI. ATP synthase, cytochrome b6f, and light-harvesting complex II proteins are detected at 60 HAI, while PSI proteins and the curvature-inducing CURT1A protein appear at 84 HAI. If stromal ribosome biogenesis is delayed, prothylakoids accumulate until stromal ribosomes are produced, and grana-forming thylakoids develop after polysomes bind to the thylakoid membranes. In fzo-like (fzl) mutants, in which thylakoid organization is perturbed, pro-grana stacks in cotyledons form discrete, spiral membrane compartments instead of organelle-wide membrane networks, suggesting that FZL is involved in fusing membrane compartments together. Our data demonstrate that the assembly of thylakoid protein complexes, CURT1 proteins, and FZL proteins mediate distinct and critical steps in thylakoid biogenesis.

A brief history of how microscopic studies led to the elucidation of the 3D architecture and macromolecular organization of higher plant thylakoids.

Microscopic studies of chloroplasts can be traced back to the year 1678 when Antonie van Leeuwenhoek reported to the Royal Society in London that he saw green globules in grass leaf cells with his single-lens microscope. Since then, microscopic studies have continued to contribute critical insights into the complex architecture of chloroplast membranes and how their structure relates to function. This review is organized into three chronological sections: During the classic light microscope period (1678-1940), the development of improved microscopes led to the identification of green grana, a colorless stroma, and a membrane envelope. More recent (1990-2020) chloroplast dynamic studies have benefited from laser confocal and 3D-structured illumination microscopy. The development of the transmission electron microscope (1940-2000) and thin sectioning techniques demonstrated that grana consist of stacks of closely appressed grana thylakoids interconnected by non-appressed stroma thylakoids. When the stroma thylakoids were shown to spiral around the grana stacks as multiple right-handed helices, it was confirmed that the membranes of a chloroplast are all interconnected. Freeze-fracture and freeze-etch methods verified the helical nature of the stroma thylakoids, while also providing precise information on how the electron transport chain and ATP synthase complexes are non-randomly distributed between grana and stroma membrane regions. The last section (2000-2020) focuses on the most recent discoveries made possible by atomic force microscopy of hydrated membranes, and electron tomography and cryo-electron tomography of cryofixed thylakoids. These investigations have provided novel insights into thylakoid architecture and plastoglobules (summarized in a new thylakoid model), while also producing molecular-scale views of grana and stroma thylakoids in which individual functional complexes can be identified.

Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell-death execution pathways downstream of ced-3 and independent of ced-9.

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1's proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.

Cis-Golgi cisternal assembly and biosynthetic activation occur sequentially in plants and algae.

The cisternal progression/maturation model of Golgi trafficking predicts that cis-Golgi cisternae are formed de novo on the cis-side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high-pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus flytrap) as determined by electron microscopy, electron tomography and immuno-electron microscopy techniques. Our findings are as follows: (i) The cis-most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3-5 COPII vesicles in contact with a C2 cis cisterna. (ii) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (iii) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (iv) C2-Cn cis cisternae grow through COPII vesicle fusion. (v) ER-resident proteins are recycled from cis cisternae to the ER via COPIa-type vesicles. (vi) In S. dubia the C2 cisternae are capable of mediating the self-assembly of scale protein complexes. (vii) In plants, ∼90% of native α-mannosidase I localizes to medial Golgi cisternae. (viii) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre-cis Golgi cisterna layer in mammalian cells.

Electron tomography of RabA4b- and PI-4Kß1-labeled trans Golgi network compartments in Arabidopsis.

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kß1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kß1/2 regulates SV size.

A three-stage model of Golgi structure and function.

The Golgi apparatus contains multiple classes of cisternae that differ in structure, composition, and function, but there is no consensus about the number and definition of these classes. A useful way to classify Golgi cisternae is according to the trafficking pathways by which the cisternae import and export components. By this criterion, we propose that Golgi cisternae can be divided into three classes that correspond to functional stages of maturation. First, cisternae at the cisternal assembly stage receive COPII vesicles from the ER and recycle components to the ER in COPI vesicles. At this stage, new cisternae are generated. Second, cisternae at the carbohydrate synthesis stage exchange material with one another via COPI vesicles. At this stage, most of the glycosylation and polysaccharide synthesis reactions occur. Third, cisternae at the carrier formation stage produce clathrin-coated vesicles and exchange material with endosomes. At this stage, biosynthetic cargo proteins are packaged into various transport carriers, and the cisternae ultimately disassemble. Discrete transitions occur as a cisterna matures from one stage to the next. Within each stage, the structure and composition of a cisterna can evolve, but the trafficking pathways remain unchanged. This model offers a unified framework for understanding the properties of the Golgi in diverse organisms.

Protein storage vacuoles are transformed into lytic vacuoles in root meristematic cells of germinating seedlings by multiple, cell type-specific mechanisms.

We have investigated the structural events associated with vacuole biogenesis in root tip cells of tobacco (Nicotiana tabacum) seedlings preserved by high-pressure freezing and freeze-substitution techniques. Our micrographs demonstrate that the lytic vacuoles (LVs) of root tip cells are derived from protein storage vacuoles (PSVs) by cell type-specific sets of transformation events. Analysis of the vacuole transformation pathways has been aided by the phytin-dependent black osmium staining of PSV luminal contents. In epidermal and outer cortex cells, the central LVs are formed by a process involving PSV fusion, storage protein degradation, and the gradual replacement of the PSV marker protein α-tonoplast intrinsic protein (TIP) with the LV marker protein γ-TIP. In contrast, in the inner cortex and vascular cylinder cells, the transformation events are more complex. During mobilization of the stored molecules, the PSV membranes collapse osmotically upon themselves, thereby squeezing the vacuolar contents into the remaining bulging vacuolar regions. The collapsed PSV membranes then differentiate into two domains: (1) vacuole "reinflation" domains that produce pre-LVs, and (2) multilamellar autophagosomal domains that are later engulfed by the pre-LVs. The multilamellar autophagosomal domains appear to originate from concentric sheets of PSV membranes that create compartments within which the cytoplasm begins to break down. Engulfment of the multilamellar autophagic vacuoles by the pre-LVs gives rise to the mature LVs. During pre-LV formation, the PSV marker α-TIP disappears and is replaced by the LV marker γ-TIP. These findings demonstrate that the central LVs of root cells arise from PSVs via cell type-specific transformation pathways.

Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography.

We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like architecture, but their size varies tremendously from approximately 15 × 30 nm to approximately 15 × 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.

3D electron tomographic and biochemical analysis of ER, Golgi and trans Golgi network membrane systems in stimulated Venus flytrap (Dionaea muscipula) glandular cells.

BACKGROUND: The insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods. RESULTS: Secretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3-6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples. CONCLUSIONS: These findings suggest that the secretory apparatus of resting gland cells is "overbuilt" to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells.

BACKGROUND: The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. RESULTS: AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50-150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. CONCLUSION: AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported antisense phenotypes of decreased gravitropic and cell enlargement responses, suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division and growth.

Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris.

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack.

Plant Cytokinesis: Terminology for Structures and Processes.

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.

Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.

Chloroplast structure: from chlorophyll granules to supra-molecular architecture of thylakoid membranes.

This review provides a brief historical account of how microscopical studies of chloroplasts have contributed to our current knowledge of the structural and functional organization of thylakoid membranes. It starts by tracing the origins of the terms plastid, grana, stroma and chloroplasts to light microscopic studies of 19th century German botanists, and then describes how different types of electron microscopical techniques have added to this field. The most notable contributions of thin section electron microscopy include the elucidation of the 3-D organization of thylakoid membranes, the discovery of prolamellar bodies in etioplasts, and the structural changes in thylakoid architecture that accompany the light-dependent transformation of etioplasts into chloroplasts. Attention is then focused on the roles that freeze-fracture and freeze-etch electron microscopy and immuno electron microscopy have played in defining the extent to which the functional complexes of thylakoids are non-randomly distributed between appressed, grana and non-appressed stroma thylakoids. Studies reporting on how this lateral differentiation can be altered experimentally, and how the spatial organization of functional complexes is affected by alterations in the light environment of plants are also included in this discussion. Finally, the review points to the possible uses of electron microscope tomography techniques in future structural studies of thylakoid membranes.

Protein disulfide isomerase-2 of Arabidopsis mediates protein folding and localizes to both the secretory pathway and nucleus, where it interacts with maternal effect embryo arrest factor.

Protein disulfide isomerase (PDI) is a thiodisulfide oxidoreductase that catalyzes the formation, reduction and rearrangement of disulfide bonds in proteins of eukaryotes. The classical PDI has a signal peptide, two CXXC-containing thioredoxin catalytic sites (a,a'), two noncatalytic thioredoxin fold domains (b,b'), an acidic domain (c) and a C-terminal endoplasmic reticulum (ER) retention signal. Although PDI resides in the ER where it mediates the folding of nascent polypeptides of the secretory pathway, we recently showed that PDI5 of Arabidopsis thaliana chaperones and inhibits cysteine proteases during trafficking to vacuoles prior to programmed cell death of the endothelium in developing seeds. Here we describe Arabidopsis PDI2, which shares a primary structure similar to that of classical PDI. Recombinant PDI2 is imported into ER-derived microsomes and complements the E. coli protein-folding mutant, dsbA. PDI2 interacted with proteins in both the ER and nucleus, including ER-resident protein folding chaperone, BiP1, and nuclear embryo transcription factor, MEE8. The PDI2-MEE8 interaction was confirmed to occur in vitro and in vivo. Transient expression of PDI2-GFP fusions in mesophyll protoplasts resulted in labeling of the ER, nucleus and vacuole. PDI2 is expressed in multiple tissues, with relatively high expression in seeds and root tips. Immunoelectron microscopy with GFP- and PDI2-specific antisera on transgenic seeds (PDI2-GFP) and wild type roots demonstrated that PDI2 was found in the secretory pathway (ER, Golgi, vacuole, cell wall) and the nuclei. Our results indicate that PDI2 mediates protein folding in the ER and has new functional roles in the nucleus.

A role of endocytosis in plant cytokinesis.

The preprophase band (PPB) of microtubules (MTs) marks the site of the future division plane irrespective of the orientation of the equatorial plane. Because the PPB MTs disappear during prometaphase, some positional information is thought to remain in the cortical cytoplasm after the disappearance of the PPB MTs. Cytoskeletal proteins are known to be excluded from the PPB site during mitosis. These depleted zones of cytoskeletal proteins are potential candidates for a "negative memory" system. However, how these depleted zones of the cytoskeletal proteins are produced remains unknown. In a recent paper, we have quantified the distribution of clathrin-coated pits and vesicles as well as of secretory structures during PPB formation using a combination of high-pressure freezing and electron tomography techniques. Our results demonstrated that the rate of endocytosis is enhanced in PPB regions. We postulate that the removal of membrane proteins by endocytosis plays a role in the creation of PPB "memory" structures.

Mitochondrial reticulation in shoot apical meristem cells of Arabidopsis provides a mechanism for homogenization of mtDNA prior to gamete formation.

Plant mitochondria are typically portrayed as being small, oval organelles. However, a recent study has demonstrated that the chondriome of shoot apical meristem (SAM) cells of Arabidopsis thaliana is unique in having two types of mitochondria, a large, central, tentaculate mitochondrion and variable numbers of small, oval mitochondria in the cell cortex that fuse with and fission from the tentaculate mitochondrion. The tentaculate mitochondrion wraps around the nucleus and persists throughout the cell cycle, undergoing distinct changes in morphology and size in a cell cycle-dependent manner. Here we demonstrate that SAM cell plastids, which also contain DNA, do not reticulate, and address the question as to why SAM cell mitochondria but not plastids form reticulate structures. We postulate that the presence of a large, tentaculate mitochondrion in SAM cells provides an efficient means for homogenizing the mitochondrial DNA and proteins during vegetative life prior to gamete production, and that this mitochondrial architecture prevents speciation. The lack of plastid reticulation in the same cells most likely reflects on the fact that the individual plastids are much larger than the small mitochondria and therefore do not need to fuse to achieve efficient intermixing of their genomes.

Statolith sedimentation kinetics and force transduction to the cortical endoplasmic reticulum in gravity-sensing Arabidopsis columella cells.

The starch statolith hypothesis of gravity sensing in plants postulates that the sedimentation of statoliths in specialized statocytes (columella cells) provides the means for converting the gravitational potential energy into a biochemical signal. We have analyzed the sedimentation kinetics of statoliths in the central S2 columella cells of Arabidopsis thaliana. The statoliths can form compact aggregates with gap sizes between statoliths approaching <30 nm. Significant intra-aggregate sliding motions of individual statoliths suggest a contribution of hydrodynamic forces to the motion of statoliths. The reorientation of the columella cells accelerates the statoliths toward the central cytoplasm within <1 s of reorientation. During the subsequent sedimentation phase, the statoliths tend to move at a distance to the cortical endoplasmic reticulum (ER) boundary and interact only transiently with the ER. Statoliths moved by laser tweezers against the ER boundary experience an elastic lift force upon release from the optical trap. High-resolution electron tomography analysis of statolith-to-ER contact sites indicate that the weight of statoliths is sufficient to locally deform the ER membranes that can potentially activate mechanosensitive ion channels. We suggest that in root columella cells, the transduction of the kinetic energy of sedimenting statoliths into a biochemical signal involves a combination of statolith-driven motion of the cytosol, statolith-induced deformation of the ER membranes, and a rapid release of kinetic energy from the ER during reorientation to activate mechanosensitive sites within the central columella cells.

ER-to-Golgi transport by COPII vesicles in Arabidopsis involves a ribosome-excluding scaffold that is transferred with the vesicles to the Golgi matrix.

Plant Golgi stacks are mobile organelles that can travel along actin filaments. How COPII (coat complex II) vesicles are transferred from endoplasmic reticulum (ER) export sites to the moving Golgi stacks is not understood. We have examined COPII vesicle transfer in high-pressure frozen/freeze-substituted plant cells by electron tomography. Formation of each COPII vesicle is accompanied by the assembly of a ribosome-excluding scaffold layer that extends approximately 40 nm beyond the COPII coat. These COPII scaffolds can attach to the cis-side of the Golgi matrix, and the COPII vesicles are then transferred to the Golgi together with their scaffolds. When Atp115-GFP, a green fluorescent protein (GFP) fusion protein of an Arabidopsis thaliana homolog of the COPII vesicle-tethering factor p115, was expressed, the GFP localized to the COPII scaffold and to the cis-side of the Golgi matrix. Time-lapse imaging of Golgi stacks in live root meristem cells demonstrated that the Golgi stacks alternate between phases of fast, linear, saltatory movements (0.9-1.25 microm/s) and slower, wiggling motions (<0.4 microm/s). In root meristem cells, approximately 70% of the Golgi stacks were connected to an ER export site via a COPII scaffold, and these stacks possessed threefold more COPII vesicles than the Golgi not associated with the ER; in columella cells, only 15% of Golgi stacks were located in the vicinity of the ER. We postulate that the COPII scaffold first binds to and then fuses with the cis-side of the Golgi matrix, transferring its enclosed COPII vesicle to the cis-Golgi.