Search

ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.Significance statementARP2/3 complex-positive dots associate exclusively with peroxisomes in plant cells, where it colocalizes with autophagosome marker ATG8f and several other proteins. Our experiments link ARP2/3 to pexophagy: colocalization between ARP2/3 dots and autophagosome increases when autophagy processes are induced or inhibited; ARP2/3 and ATG8f colocalize and co-immunoprecipitate, and finally, ARP2/3 mutants’ cells contain more peroxisomes than WT. Our results suggest a novel role of ARP2/3 in peroxisome structure and function regulation.

Plant cell organelles are highly mobile and their positioning play key roles in plant growth, development and responses to changing environmental conditions. Movement is acto-myosin dependent. Despite controlling the dynamics of several organelles, myosin and myosin receptors identified so far in Arabidopsis thaliana generally do not localise to the organelles whose movement they control, raising the issue of how specificity is determined. Here we show that a MyoB myosin receptor, MRF7, specifically localises to the Golgi membrane and affects its movement. Myosin XI-K was identified as a putative MRF7 interactor through mass spectrometry analysis. Co-expression of MRF7 and XI-K tail triggers the relocation of XI-K to the Golgi, linking a MyoB/myosin complex to a specific organelle in Arabidopsis. FRET-FLIM confirmed the in vivo interaction between MRF7 and XI-K tail on the Golgi and in the cytosol, suggesting that myosin/myosin receptor complexes perhaps cycle on and off organelle membranes. This work supports a traditional mechanism for organelle movement where myosins bind to receptors and adaptors on the organelle membranes, allowing them to actively move on the actin cytoskeleton, rather than passively in the recently proposed cytoplasmic streaming model., Perico et al. use co-expression analysis and a FRET-FLIM approach to show that the Arabidopsis MyoB myosin receptor, MRF7, triggers the relocation of Myosin XI-K to the Golgi. As such, this study provides evidence for plant myosin recruitment and control of organelle movement.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions. Fil: Savage, Zachary. Imperial College London; Reino Unido Fil: Duggan, Cian. Imperial College London; Reino Unido Fil: Toufexi, Alexia. Imperial College London; Reino Unido Fil: Pandey, Pooja. Imperial College London; Reino Unido Fil: Liang, Yuxi. Imperial College London; Reino Unido Fil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Yuen, Lok Him. Imperial College London; Reino Unido Fil: Gaboriau, David C. A.. Imperial College London; Reino Unido Fil: Leary, Alexandre Y.. Imperial College London; Reino Unido Fil: Tumtas, Yasin. Imperial College London; Reino Unido Fil: Khandare, Virendrasinh. Imperial College London; Reino Unido Fil: Ward, Andrew D.. Science and Technology Facilities Council; Reino Unido Fil: Botchway, Stanley W.. Science and Technology Facilities Council; Reino Unido Fil: Bateman, Benji C.. Science and Technology Facilities Council; Reino Unido Fil: Pan, Indranil. Alan Turing Institute; Reino Unido. Imperial College London; Reino Unido Fil: Schattat, Martin. Martin Luther Universitat Halle-Wittenberg; Alemania Fil: Sparkes, Imogen. University of Bristol; Reino Unido Fil: Bozkurt, Osman Tolga. Imperial College London; Reino Unido

The plant endoplasmic reticulum (ER) defines the biosynthetic site of lipids and proteins destined for secretion, but also contains important signal transduction and homeostasis components that regulate multiple hormonal and developmental responses. To achieve its various functions, the ER has a unique architecture, both reticulated and highly plastic, that facilitates the spatial-temporal segregation of biochemical reactions and the establishment of inter-organelle communication networks. At the cell cortex, the cortical ER (cER) anchors to and functionally couples with the PM through largely static structures known as ER-PM contact sites (EPCS). These spatially confined microdomains are emerging as critical regulators of the geometry of the cER network, and as highly specialized signalling hubs. In this review, we share recent insights into how EPCS regulate cER remodelling, and discuss the proposed roles for plant EPCS components in the integration of environmental and developmental signals at the cER-PM interface.

The endoplasmic reticulum (ER) in plant cells forms a highly dynamic network of complex geometry. ER network morphology and dynamics are influenced by a number of biophysical processes, including filament/tubule tension, viscous forces, Brownian diffusion, and interactions with many other organelles and cytoskeletal elements. Previous studies have indicated that ER networks can be thought of as constrained minimal-length networks acted on by a variety of forces that perturb and/or remodel the network. Here, we study two specific biophysical processes involved in remodeling. One is the dynamic relaxation process involving a combination of tubule tension and viscous forces. The other is the rapid creation of cross-connection tubules by direct or indirect interactions with cytoskeletal elements. These processes are able to remodel the ER network: the first reduces network length and complexity whereas the second increases both. Using live cell imaging of ER network dynamics in tobacco leaf epidermal cells, we examine these processes on ER network dynamics. Away from regions of cytoplasmic streaming, we suggest that the dynamic network structure is a balance between the two processes, and we build an integrative model of the two processes for network remodeling. This model produces quantitatively similar ER networks to those observed in experiments. We use the model to explore the effect of parameter variation on statistical properties of the ER network.

Mitochondria are highly pleomorphic, undergoing rounds of fission and fusion. Mitochondria are essential for energy conversion, with fusion favouring higher energy demand. Unlike fission, the molecular components involved in mitochondrial fusion in plants are unknown. Here, we show a role for the GTPase Miro2 in mitochondria interaction with the ER and its impacts on mitochondria fusion and motility. Mutations in AtMiro2’s GTPase domain indicate that the active variant results in larger, fewer mitochondria which are attached more readily to the ER when compared with the inactive variant. These results are contrary to those in metazoans where Miro predominantly controls mitochondrial motility, with additional GTPases affecting fusion. Synthetically controlling mitochondrial fusion rates could fundamentally change plant physiology by altering the energy status of the cell. Furthermore, altering tethering to the ER could have profound effects on subcellular communication through altering the exchange required for pathogen defence., White et al show using quantitative imaging and optical tweezers that in tobacco leaf cells, the Miro2 GTPase promotes mitochondrial fusion and attachment to the ER. This is in contrast to metazoans where Miro mainly controls mitochondrial motility, with additional GTPases affecting fusion.

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterised. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalyzing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual targeted proteins in the classic sense of having targeting sequences that directde novosynthesised proteins to specific cellular locations. Accumulating evidence suggests that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox associated enzymes.

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.

• The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant specific NETWORKED protein, NET3C, and phylogenetically conserved Vesicle-Associated Membrane Protein-Associated Proteins (VAPs). • Ten VAP homologues (VAP27-1 to 10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade have been tagged with fluorescent protein and expressed in Nicotiana benthamiana. • Proteins from clades one and three localised to the ER as well as to ER/PM contact sites (EPCS), whereas proteins from clade two are found only at the PM. Some of the VAP27 labelled EPCS localised to plasmodesmata, and we show that the mobility of VAP27 at the EPCS is influenced by the cell wall. EPCS closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. • VAP27 labelled EPCS are found in most cell types in Arabidopsis with the exception of cells in early trichome development. Arabidopsis expressing VAP27-GFP fusions exhibit pleiotropic phenotypes including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. • Taken together these data indicate that VAP27 proteins used at the EPCS are essential for normal ER-cytoskeleton interaction and for plant development.

Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.

Optical tweezers enable users to physically trap organelles and move them laterally within the plant cell. Recent advances have highlighted physical interactions between functionally related organelle pairs, such as ER–Golgi and peroxisome–chloroplast, and have shown how organelle positioning affects plant growth. Quantification of these processes has provided insight into the force components which ultimately drive organelle movement and positioning in plant cells. Application of optical tweezers has therefore revolutionised our understanding of plant organelle dynamics.

Contents Summary 381 I. Introduction 381 II. Basic movement characteristics 382 III. Actin and associated motors, myosins, play a primary role in plant organelle movement and positioning 382 IV. Mechanisms of myosin recruitment: a tightly regulated system? 384 V. Microtubules, associated motors and interplay with actin 386 VI. Role of organelle interactions: tales of tethers 387 VII. Summary model to describe organelle movement in higher plants 390 VIII. Why is organelle movement important? 390 IX. Conclusions and future perspectives 391 Acknowledgements 391 References 391 SUMMARY: Organelle movement and positioning are correlated with plant growth and development. Movement characteristics are seemingly erratic yet respond to external stimuli including pathogens and light. Given these clear correlations, we still do not understand the specific roles that movement plays in these processes. There are few exceptions including organelle inheritance during cell division and photorelocation of chloroplasts to prevent photodamage. The molecular and biophysical components that drive movement can be broken down into cytoskeletal components, motor proteins and tethers, which allow organelles to physically interact with one another. Our understanding of these components and concepts has exploded over the past decade, with recent technological advances allowing an even more in-depth profiling. Here, we provide an overview of the cytoskeletal and tethering components and discuss the mechanisms behind organelle movement in higher plants.

Optical tweezers have been used to trap and micromanipulate several biological specimens ranging from DNA, macromolecules, organelles to single celled organisms. Using a combination of the refraction and scattering of laser light from a focused laser beam, refractile objects are physically captured and can be moved within the surrounding media. The technique is routinely used to determine biophysical properties such as the forces exerted by motor proteins. Here, we describe how optical tweezers combined with total internal reflection fluorescence (TIRF) microscopy can be used to assess physical interactions between organelles, more specifically the ER and Golgi bodies in plant cells.

The endoplasmic reticulum (ER) in live cells is a highly mobile network whose structure dynamically changes on a number of timescales. The role of such drastic changes in any system is unclear, although there are correlations with ER function. A better understanding of the fundamental biophysical constraints on the system will allow biologists to determine the effects of molecular factors on ER dynamics. Previous studies have identified potential static elements that the ER may remodel around. Here, we use these structural elements to assess biophysical principles behind the network dynamics. By analyzing imaging data of tobacco leaf epidermal cells under two different conditions, i.e., native state (control) and latrunculin B (treated), we show that the geometric structure and dynamics of ER networks can be understood in terms of minimal networks. Our results show that the ER network is well modeled as a locally minimal-length network between the static elements that potentially anchor the ER to the cell cortex over longer timescales; this network is perturbed by a mixture of random and deterministic forces. The network need not have globally minimum length; we observe cases where the local topology may change dynamically between different Euclidean Steiner network topologies. The networks in the treated cells are easier to quantify, because they are less dynamic (the treatment suppresses actin dynamics), but the same general features are found in control cells. Using a Langevin approach, we model the dynamics of the nonpersistent nodes and use this to show that the images can be used to estimate both local viscoelastic behavior of the cytoplasm and filament tension in the ER network. This means we can explain several aspects of the ER geometry in terms of biophysical principles.

Several ureides are intermediates of purine base catabolism, releasing nitrogen from the purine nucleotides for reassimilation into amino acids. In some legumes like soybean (Glycine max), ureides are used for nodule-to-shoot translocation of fixed nitrogen. Four enzymes of Arabidopsis (Arabidopsis thaliana), (1) allantoinase, (2) allantoate amidohydrolase (AAH), (3) ureidoglycine aminohydrolase, and (4) ureidoglycolate amidohydrolase (UAH), catalyze the complete hydrolysis of the ureide allantoin in vitro. However, the metabolic route in vivo remains controversial. Here, in growth and metabolite analyses of Arabidopsis mutants, we demonstrate that these enzymes are required for allantoin degradation in vivo. Orthologous enzymes are present in soybean, encoded by one to four gene copies. All isoenzymes are active in vitro, while some may be inefficiently translated in vivo. Surprisingly, transcript and protein amounts are not significantly regulated by nitrogen fixation or leaf ureide content. A requirement for soybean AAH and UAH for ureide catabolism in leaves has been demonstrated by the use of virus-induced gene silencing. Functional AAH, ureidoglycine aminohydrolase, and UAH are also present in rice (Oryza sativa), and orthologous genes occur in all other plant genomes sequenced to date, indicating that the amidohydrolase route of ureide degradation is universal in plants, including mosses (e.g. Physcomitrella patens) and algae (e.g. Chlamydomomas reinhardtii).

The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER), and synthesises cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role of cis-Golgi plant golgins (long coiled-coil domains proteins anchored to Golgi membranes) in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap, and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC, and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.HighlightHere we show that the Golgi-associated Arabidopsis thaliana protein AtCASP may form part of a golgin-mediated tethering complex involved in anchoring plant Golgi stacks to the endoplasmic reticulum (ER).

Without doubt, GFP and spectral derivatives have revolutionized the way biologists approach their journey toward the discovery of how plant cells function. It is fascinating that in its early days GFP was used merely for localization studies, but as time progressed researchers successfully explored new avenues to push the power of GFP technology to reach new and exciting research frontiers. This has had a profound impact on the way we can now study complex and dynamic systems such as plant endomembranes. Here we briefly describe some of the approaches where GFP has revolutionized in vivo studies of protein distribution and dynamics and focus on two emerging approaches for the application of GFP technology in plant endomembranes, namely optical tweezers and forward genetics approaches, which are based either on the light or on genetic manipulation of secretory organelles to gain insights on the factors that control their activities and integrity.

We have identified two endoplasmic reticulum (ER)-associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over-expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox-based topology assay (ReTA) with redox-sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N- and C-termini were found on the cytosolic side of the ER. A C-terminal di(tri)-lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP-KMS1 signal in the ER. Over-expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked-down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.

Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape the ER into tubules. It has been hypothesized that their unusually long conserved hydrophobic regions cause reticulons to assume a wedge-like topology that induces membrane curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana reticulon (RTNLB13) localized to, and induced constrictions in, cortical ER tubules. Ectopic expression of RTNLB13 was sufficient to induce ER tubulation in an Arabidopsis mutant (pah1 pah2) whose ER membrane is mostly present in a sheet-like form. By sequential shortening of the four transmembrane domains (TMDs) of RTNLB13, we show that the length of the transmembrane regions is directly correlated with the ability of RTNLB13 to induce membrane tubulation and to form low-mobility complexes within the ER membrane. We also show that full-length TMDs are necessary for the ability of RTNLB13 to reside in the ER membrane.

Organelle movement in plants cells is extremely dynamic. Movement is driven by the acto-myosin system. Higher plant myosins fall into two classes: classes XI and VIII. Localization studies have highlighted that myosins are present throughout the cytosol, label motile puncta and decorate the nuclear envelope and plasma membrane. Functional studies through expression of dominant-negative myosin variants, RNAi (RNA interference) and T-DNA insertional analysis have shown that class XI myosins are required for organelle movement. Intriguingly, organelle movement is also linked to Arabidopsis growth and development. The present review tackles current findings relating to plant organelle movement and the role of myosins.

Plant peroxisomes are extremely dynamic, moving and undergoing changes of shape in response to metabolic and environmental signals. Matrix proteins are imported via one of two import pathways, depending on the targeting signal within the protein. Each pathway has a specific receptor but utilizes common membrane-bound translocation machinery. Current models invoke receptor recycling, which may involve cycles of ubiquitination. Some components of the import machinery may also play a role in proteolytic turnover of matrix proteins, prompting parallels with the endoplasmic-reticulum-associated degradation pathway. Peroxisome membrane proteins, some of which are imported post-translationally, others of which may traffic to peroxisomes via the endoplasmic reticulum, use distinct proteinaceous machinery. The isolation of mutants defective in peroxisome biogenesis has served to emphasize the important role of peroxisomes at all stages of the plant life cycle.

This work was supported by grant BB/J004987/1 from the British Biotechnology and Biological Sciences Research Council (BBSRC) to K.O and C.H and by The Leverhulme Trust (F/00 382/G) to C.H. Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell plate formation. Here, we show that members of the reticulon (RTNLB) family of ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play a role in the formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its center, where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation. Postprint

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.

International audience; The endoplasmic reticulum (ER) is an intricate network that pervades the entire cortex of plant cells and its geometric shape undergoes drastic changes. This paper proposes a mathematical model to reconstruct geometric network dynamics by combining the node movements within the network and topological changes engendered by these nodes. The network topology in the model is determined by a modified optimization procedure from the work (Lemarchand, et. al. 2014) which minimizes the total length taking into account both degree and angle constraints, beyond the conditions of connectedness and planarity. A novel feature for solving our optimization problem is the use of ’lifted’ angle constraints, which allows one to considerably reduce the solution runtimes. Using this optimization technique and a Langevin approach for the branching node movement, the simulated network dynamics represent the ER network dynamics observed under latrunculin B treated condition and recaptures features such as the appearance/disappearance of loops within the ER under the native condition. The proposed modeling approach allows quantitative comparison of networks between the model and experimental data based on topological changes induced by node dynamics. An increased temporal resolution of experimental data will allow a more detailed comparison of network dynamics using this modeling approach.

The endoplasmic reticulum (ER) of higher plants is a complex network of tubules and cisternae. Some of the tubules and cisternae are relatively persistent, while others are dynamically moving and remodelling through growth and shrinkage, cycles of tubule elongation and retraction, and cisternal expansion and diminution. Previous work showed that transient expression in tobacco leaves of the motor-less, truncated tail of myosin XI-K increases the relative area of both persistent cisternae and tubules in the ER. Likewise, transient expression of XI-K tail diminishes the movement of organelles such as Golgi and peroxisomes. To examine whether other class XI myosins are involved in the remodelling and movement of the ER, other myosin XIs implicated in organelle movement, XI-1 (MYA1),XI-2 (MYA2), XI-C, XI-E, XI-I, and one not, XI-A, were expressed as motor-less tail constructs and their effect on ER persistent structures determined. Here, we indicate a differential effect on ER dynamics whereby certain class XI myosins may have more influence over controlling cisternalisation rather than tubulation.

Peroxisomes are eukaryotic organelles that perform diverse and variable functions. Although genetic studies in yeasts and mammals have identified approximately 20 genes (PEX genes) required for the biogenesis of this important organelle, biochemical studies of protein targeting and import have lagged behind and in many cases we have no idea of the function of the PEX gene products (peroxins). Using an import assay in vitro derived from sunflower cotyledon cells and recombinant proteins, we have obtained translocation intermediates on the peroxisome import pathway and are using cross-linking to identify interacting partners. We have also used antibodies raised against human PEX14 to inhibit the import of matrix proteins in this system. To obtain homologous antibodies for inhibition experiments, to immunoprecipitate cross-linked products and to enable us to study the import pathways of peroxins we have cloned and characterized plant orthologues of three PEX genes, PEX6, PEX10 and PEX14.

SummaryThe cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants [1, 2], and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins [3], (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein [4, 5], and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23 mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2-, whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology., DP and MR-S acknowledge fellowships JAE-Pre and JAE-DOC, respectively, from the CSIC, and the European Social Fund (ESF). This work was supported by ERDF-co-financed grants BIO2008-04067 and BIO2012-36742 from MICINN and Junta de Andalucía (BIO-337), Spain. The authors acknowledge Dr D. McCurdy and Dr Sagi for providing the GFP–FABD2 and xdh Arabidopsis lines, respectively. We thank Dr T. Ketelaar for critical reading of the manuscript. The confocal laser fluorescence microscopy analyses were carried out at the Technical Services of the University of Jaén and University of Granada.

Compartmentalization of metabolic functions in membrane bounded organelles is a defining characteristic of eukaryotes. Movement, positioning and morphology of such organelles are key determinants for function, maintenance and inheritance. For example, mutations in the molecular motors (myosin) that drive organelle movement in plants result in dwarf plants with reduced seed set. Therefore, movement and positioning of organelles are key factors for plant development and growth.

16p; International audience; We have studied the network geometry of the endoplasmic reticulum by means of graph theoretical and integer programming models. The purpose is to represent this structure as close as possible by a class of finite, undirected and connected graphs the nodes of which have to be either of degree three or at most of degree three. We determine plane graphs of minimal total edge length satisfying degree and angle constraints, and we show that the optimal graphs are close to the ER network geometry. Basically, two procedures are formulated to solve the optimization problem: a binary linear program, that iteratively constructs an optimal solution, and a linear program, that iteratively exploits additional cutting planes from different families to accelerate the solution process. All formulations have been implemented and tested on a series of real-life and randomly generated cases. The cutting plane approach turns out to be particularly efficient for the real-life testcases, since it outperforms the pure integer programming approach by a factor of at least 10.

The plant endoplasmic reticulum (ER) is responsible for the synthesis and often storage of a large group of proteins and lipids that enter the secretory pathway. This multifunctional organelle, which also represents one of the calcium storage compartments in plant cells, has currently received considerable attention from the research community because of features unique to plants that make it particularly interesting for biotechnology. Here, the principles behind ER dynamics in plants, and the molecular factors that control the rapid remodelling and network configuration of the organelle are discussed. Correlations between ER morphology and function indicate the potential to enhance protein storage by merely increasing the capacity of the organelle through altering its shape. Key Concepts: The endoplasmic reticulum in plants rapidly changes ‘shape’. The ER is composed of a polygonal network of tubules and cisternae that readily interconvert. The ER membrane shaping proteins, reticulons, affect ER curvature whereas root hair defective 3 (RHD3) may affect tubule fusion. ER network movement is driven by actin and myosin in plants. The ER is physically connected to Golgi bodies in plant cells. Keywords: endoplasmic reticulum; Golgi bodies; network remodelling; movement; reticulons; RHD3

Without doubt, GFP and spectral derivatives have revolutionized the way biologists approach their journey toward the discovery of how plant cells function. It is fascinating that in its early days GFP was used merely for localization studies, but as time progressed researchers successfully explored new avenues to push the power of GFP technology to reach new and exciting research frontiers. This has had a profound impact on the way we can now study complex and dynamic systems such as plant endomembranes. Here we briefly describe some of the approaches where GFP has revolutionized in vivo studies of protein distribution and dynamics and focus on two emerging approaches for the application of GFP technology in plant endomembranes, namely optical tweezers and forward genetics approaches, which are based either on the light or on genetic manipulation of secretory organelles to gain insights on the factors that control their activities and integrity.

Summary The endoplasmic reticulum (ER) is a network of membrane sheets and tubules connected via three-way junctions. A family of proteins, the reticulons, are responsible for shaping the tubular ER. Reticulons interact with other tubule-forming proteins (Dp1 and Yop1p) and the GTPase atlastin. The Arabidopsis homologue of Dp1/Yop1p is HVA22. We show here that a seed-specific isoform of HVA22 labels the ER in tobacco (Nicotiana tabacum) cells but its overexpression does not alter ER morphology. The closest plant homologue of atlastin is RHD3. We show that RHD3-like 2 (RL2), the seed-specific isoform of RHD3, locates to the ER without affecting its shape or Golgi mobility. Expression of RL2-bearing mutations within its GTPase domain induces the formation of large ER strands, suggesting that a functional GTPase domain is important for the formation of three-way junctions. Coexpression of the reticulon RTNLB13 with RL2 resulted in a dramatic alteration of the ER network. This alteration did not depend on an active GTPase domain but required a functional reticulon, as no effect on ER morphology was seen when RL2 was coexpressed with a nonfunctional RTNLB13. RL2 and its GTPase mutants coimmunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 act together in modulating ER morphology.

Plant myosins are required for organelle movement, and a role in actin organization has recently come to light. Myosin mutants display several gross morphological phenotypes, the most severe being dwarfism and reduced fecundity, and there is a correlation between reduced organelle movement and morphological defects. This review aims to discuss recent findings in plants relating to the role of myosins in actin dynamics, development, and organelle movement, more specifically the endoplasmic reticulum (ER). One overarching theme is that there still appear to be more questions than answers relating to plant myosin function and regulation.

The endoplasmic reticulum (ER) in higher plants performs many important functions, yet our understanding of how its intricate network shape and dynamics relate to function is very limited. Recent work has begun to unpick key molecular players in the generation of the pleomorphic, highly dynamic ER network structure that pervades the entire cytoplasm. ER movement is acto-myosin dependent. ER shape is dependent on RHD3 (Root Hair Defective 3) and a family of proteins called reticulons. The major challenge that lies ahead is understanding how factors that control ER shape and movement are regulated and how this relates to the numerous functions of the ER.

We have identified two endoplasmic reticulum (ER)-associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over-expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox-based topology assay (ReTA) with redox-sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N- and C-termini were found on the cytosolic side of the ER. A C-terminal di(tri)-lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP-KMS1 signal in the ER. Over-expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked-down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.

International audience; Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat gamma-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that gamma-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in gamma-gliadin ER retention and PBLS formation. The high actin-dependent mobility of gamma-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both gamma-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to gamma-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP 'core'. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions.

Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape the ER into tubules. It has been hypothesized that their unusually long conserved hydrophobic regions cause reticulons to assume a wedge-like topology that induces membrane curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana reticulon (RTNLB13) localized to, and induced constrictions in, cortical ER tubules. Ectopic expression of RTNLB13 was sufficient to induce ER tubulation in an Arabidopsis mutant (pah1 pah2) whose ER membrane is mostly present in a sheet-like form. By sequential shortening of the four transmembrane domains (TMDs) of RTNLB13, we show that the length of the transmembrane regions is directly correlated with the ability of RTNLB13 to induce membrane tubulation and to form low-mobility complexes within the ER membrane. We also show that full-length TMDs are necessary for the ability of RTNLB13 to reside in the ER membrane.

Laser tweezers, often known as optical tweezers or optical traps, permit the capturing and micromanipulation of microscopic particles along X, Y and Z axes using the radiation pressure generated by a focused laser beam, normally in the infrared region of the spectrum. For trapping to be successful, the object to be captured must have a higher refractive index than that of its surrounding medium and forces generated by individual traps must be in the piconewton range [1]. Single optical traps are generally used to capture particles in the micron range, although multiple traps have been developed that can be utilized to move larger objects. Optical traps are commonly used in single-molecule techniques where, for example, they can be used to measure forces exerted on polymer beads coated with motor proteins. Thus, for instance, the mechanism of kinesin walking on microtubules or myosin on actin can be probed [[2] and [3]]. At the other end of the spectrum the optical traps are often used for the mechanical stimulation of cells [4] or manipulation of whole cells such as germinating fungal conidia [5]. What is, however, very apparent is that the full potential for using optical tweezers to manipulate organelles within living cells has yet to be fully exploited, with the majority of reports working at the whole-cell or single-molecule levels. Ashkin and Dziedzic [6•] were perhaps the first to show that cytoplasmic particles can be manipulated in vivo, when they demonstrated the pulling cytoplasm strands across vacuoles of onion epidermal cells and displacement of Spirogyra chloroplasts. Redirection of the growth of fungal hyphae by manipulation of the Spitzenkörper has been elegantly demonstrated through lateral displacement of this tip organelle [7]. Recently, and often in combination with confocal microscopy, it has been demonstrated that optical traps can be a very powerful tool in unravelling cytoplasmic dynamics. Here we discuss some of the few applications that have revealed new physical aspects of cytoplasmic and organelle dynamics in plant cells.

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.

The PEX11 family of peroxisome membrane proteins have been shown to be involved in regulation of peroxisome size and number in plant, animals, and yeast cells. We and others have previously suggested that peroxisome proliferation as a result of abiotic stress may be important in plant stress responses, and recently it was reported that several rice PEX11 genes were up regulated in response to abiotic stress. We sought to test the hypothesis that promoting peroxisome proliferation in Arabidopsis thaliana by over expression of one PEX11 family member, PEX11e, would give increased resistance to salt stress. We could demonstrate up regulation of PEX11e by salt stress and increased peroxisome number by both PEX11e over expression and salt stress, however our experiments failed to find a correlation between PEX11e over expression and increased peroxisome metabolic activity or resistance to salt stress. This suggests that although peroxisome proliferation may be a consequence of salt stress, it does not affect the ability of Arabidopsis plants to tolerate saline conditions.

Using a novel analytical tool, this study investigates the relative roles of actin, microtubules, myosin, and Golgi bodies on form and movement of the endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) leaf epidermal cells. Expression of a subset of truncated class XI myosins, which interfere with the activity of native class XI myosins, and drug-induced actin depolymerization produce a more persistent network of ER tubules and larger persistent cisternae. The treatments differentially affect two persistent size classes of cortical ER cisternae, those >0.3 μm2 and those smaller, called punctae. The punctae are not Golgi, and ER remodeling occurs in the absence of Golgi bodies. The treatments diminish the mobile fraction of ER membrane proteins but not the diffusive flow of mobile membrane proteins. The results support a model whereby ER network remodeling is coupled to the directionality but not the magnitude of membrane surface flow, and the punctae are network nodes that act as foci of actin polymerization, regulating network remodeling through exploratory tubule growth and myosin-mediated shrinkage.

The ER (endoplasmic reticulum) in higher plants forms a pleomorphic web of membrane tubules and small cisternae that pervade the cytoplasm, but in particular form a polygonal network at the cortex of the cell which may be anchored to the plasma membrane. The network is associated with the actin cytoskeleton and demonstrates extensive mobility, which is most likely to be dependent on myosin motors. The ER is characterized by a number of domains which may be associated with specific functions such as protein storage, or with direct interaction with other organelles such as the Golgi apparatus, peroxisomes and plastids. In the present review we discuss the nature of the network, the role of shape-forming molecules such as the recently described reticulon family of proteins and the function of some of the major domains within the ER network.

Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.

The plant Golgi apparatus (GA) as its counterparts in mammalian, insect and fungal cells is a multifunctional organelle not only receiving and modifying cargo delivered from the endoplasmic reticulum (ER) for export, but also synthesising lipids and many of the complex polysaccharides of the cell wall (Neumann et al. 2003). It is also likely that the organelle acts as one of several destinations for endocytosed material (Fowke et al. 1991). The GA in a plant cell is composed of numerous, sometimes many hundreds, individual stacks of cisternae being approximately 1 mm in diameter (Fig. 1). These superficially resemble the stacks reported within insect cells such as Drosophila (Kondylis et al. 2005), with the major difference in that in elongate or mature cells individual stacks demonstrate an actin-based motility, showing a range of movements associated with the surface of ER tubules (Boevink et al. 1998). In this chapter we will concentrate on some of the features that make the plant Golgi unique, such as the motility, the interface with the ER and mechanisms of transport within and from the stack.

Peroxisomes are organelles with an essentially oxidative metabolism that are involved in various metabolic pathways such as fatty acid beta-oxidation, photorespiration, and metabolism of reactive oxygen species (ROS) and reactive nitrogen species. These organelles are highly dynamic but there is little information about the regulation of, and the effects of environment on, peroxisome movement. In this work a stable Arabidopsis line expressing the GFP-SKL peptide targeted to peroxisomes was characterized. Peroxisome-associated fluorescence was observed in all tissues, including leaves (mesophyll and epidermal cells, trichomes, and stomata) and roots. The dynamics of peroxisomes in epidermal cells was examined by confocal laser microscope, and various types of movement were observed. The speed of movement differed depending on the plant age. Treatment of plants with CdCl(2) (100 microM) produced a significant increase in speed, which was dependent on endogenous ROS and Ca(2+), but was not related to actin cytoskeleton modifications. In light of the results obtained, it is proposed that the increase in peroxisomal motility observed in Arabidopsis plants could be a cellular mechanism of protection against the Cd-imposed oxidative stress. Other possible roles for the enhanced peroxisome movement in plant cell physiology are discussed.