The endoplasmic reticulum (ER) is a dynamic subcellular compartment that is essential to eukaryotic life because it contributes significantly to the synthesis of fundamental building blocks of the cell, including proteins and lipids, and it acts as an important architectural scaffold to maintain a well-organized spatial distribution of the other endomembrane organelles. Recent analyses with live cell imaging coupled with genetics studies have brought to light the incredible dynamism of this organelle and the underlying drivers as well as the impact of the ER organization on the general cellular homeostasis and plant growth. In this review, we highlight the most recent advances in the understanding of the mechanisms that enable the morphological integrity of the plant ER in relation to the other organelles and the cytoskeleton. At a submicron level, the ER network is organized in domains that are morphologically distinct and that assume specific functions (Staehelin, 1997). These characteristics make the ER a morphologically continuous cellular compartment that is nonuniform at the functional and structural level. In humans, alterations in ER-mediated processes cause disease phenotypes that have been classified into three groups: (1) cargo retention and degradation, (2) cargo accumulation and ER stress, and (3) ER transport machinery diseases (Aridor and Hannan, 2000). Also in plant cells, defects in ER functionality lead to various developmental defects (Tamura et al., 2005; Conger et al., 2011; Stefano et al., 2012; Renna et al., 2013), supporting a critical role of the ER for organism biology at large. Morphologically, the ER is able to reorganize, enlarge, and contract its highly dynamic polygonal tubular network both spatially and temporally (Ridge et al., 1999; Sparkes et al., 2009a; Stefano et al., 2014). The integrity of the ER network structure is important to maintain an efficient unfolded protein response (UPR), as demonstrated by the evidence that loss of ER-shaping proteins leads to attenuation of the UPR signaling in conditions of accrual of unfolded secretory proteins in the ER (Lai et al., 2014). Therefore, there is a strong connection between the morphology and the functional integrity of the ER. The plant ER also entertains strong functional connections with other organelles, including the Golgi apparatus and the vacuole to which newly synthesized proteins can be exported (Brandizzi et al., 2002; Shimada et al., 2003; Brandizzi, 2017), but also with chloroplasts with which the ER synthesizes essential lipids (Hurlock et al., 2014; Block and Jouhet, 2015). Possibly via direct connections, the ER movement influences the movement of other organelles, supporting an emerging model in cell biology that the ER interaction with other organelles is important not only for the exchange of constituents with other cellular compartments but also for their spatial distribution and function (Stefano et al., 2014, 2015) In this review, we highlight some of the recent and exciting literature addressing the fundamental questions on how the ER morphology and dynamics are controlled and how the ER interacts with the cytoskeleton and other organelles in plant cells. In general, the plant ER assumes the shape of a membrane network resembling the arrangement of a spider web with interconnected tubules and cisternae within the cell. Although the bulk of the ER is restricted at the cell cortex where it is sandwiched between the tonoplast and the plasma membrane (PM), long ER tubular strands characterized by a high streaming velocity cross along the central vacuole (Ueda et al., 2010; Sparkes et al., 2011). Electron microscopy studies have revealed the presence of smooth ER, rough ER, and nuclear envelope regions (Hawes et al., 1981; Craig and Staehelin, 1988; Staehelin, 1997). The rough and smooth ER regions are subdomains with associated ribosomes or ribosome-free regions, respectively. The nuclear envelope is enwrapped by the ER, resulting in a double membrane delimiting the nucleus. Additionally, the ER passes through the plasmodesmata, which are tiny channels that protrude into the cell wall and interconnect the cytoplasm of neighboring cells (Carr, 1976; Wright and Oparka, 2006). These structures are unique to plants and are crossed by a narrow tube-like structure, named desmotubule, which is derived from the ER (Quader and Zachariadis, 2006; Knox et al., 2015; Nicolas et al., 2017). As a result, the ER of each cell is interconnected to the neighboring cells through these channels, forming a virtually unique organelle whose extension is not delimited by the cell’s boundaries. The ER also is attached to the PM through ER-PM contact sites (EPCSs), which are largely immotile subdomains of the ER that underlie the PM and whose number and density at the cell cortex diminish as cells expand (McFarlane et al., 2017). Fusion profiles of the ER membrane with the PM at the EPCSs have not been observed; however, proteins such as VAP27 proteins and Synaptotagmin1 (SYT1) have been shown to accumulate at the EPCSs (Wang et al., 2014; Levy et al., 2015; Pérez-Sancho et al., 2015; Siao et al., 2016; McFarlane et al., 2017). The VAP proteins are conserved across kingdoms and possess three main regions, a C-terminal transmembrane domain, an N-terminal major sperm domain, and a coiled-coil domain (Wang et al., 2014). The VAP subfamily of VAP27 proteins may contribute to ER anchoring to the cell surface via the plasmodesmata as well as bridging the ER with the PM, through yet unknown mechanisms that depend on cell wall integrity (Wang P. et al., 2016). The plant SYT1 is a close ortholog of the yeast tricalbins and metazoan synaptotagmins that serve as ER-PM anchors in these nonplant cell systems. Similar to VAP27, SYT1 is localized to the bulk ER, and the EPCSs and SYT1 has been localized at EPCSs demarcated by VAP27 (Pérez-Sancho et al., 2015). Based on the ability of VAP27 to interact with the plant-specific actin-binding protein NET3c and microtubules (MTs), it appears that the EPCSs marked by VAP27 may serve as ER-PM hubs where the two major cytoskeletal components of plant cells converge (Wang et al., 2014). While roles for nonplant EPCSs are emerging, including lipid homeostasis and Ca2+ influx (van der Kant and Neefjes, 2014; Wakana et al., 2015), a physiological role for the plant EPCSs has yet to be defined. It has been demonstrated that SYT1 interacts with phospholipids possibly through electrostatic interactions and may therefore bridge the ER membrane with the PM. A loss of SYT1 reduces the ability of cells to withstand mechanical pressure (Pérez-Sancho et al., 2015), which may underlie a function of these sites in the plant adaptation to abiotic stresses present in the natural environment. The molecular mechanisms underlying such a role have yet to be explored, but the evidence that the EPCSs demarcated by SYT1 largely overlap with VAP27-EPCSs suggests a functional connection between mechanosensing and cytoskeletal organization at these enigmatic sites. The identification of SYT1 and VAP27 as components of the EPCS proteome opens up the exciting opportunity to further define the EPCS constituents, which may provide additional tools to understand the cellular role(s) of plant EPCSs. With electron microscopy analyses, in addition to the EPCSs and plasmodesmata, the plant ER also has been found in contact with other membranes, including Golgi, mitochondria, vacuole, and plastids (Juniper et al., 1982; Staehelin, 1997). Indeed, using laser trap technology, it has been shown that the ER physically contacts the chloroplasts obtained from ruptured protoplasts expressing a fluorescent ER marker. More specifically, it was shown that the released chloroplasts remained attached to ER fragments that could be stretched out by optical tweezers. The applied force of 400 pN, which is a magnitude compatible with protein-protein interactions, could not drag a chloroplast free from its attached ER (Andersson et al., 2007). Using confocal imaging of a fluorescent fusion, the Brassica napus CLIP1 lipase/acylhydrolase (BnCLIP1) has been detected recently in transient expression in tobacco (Nicotiana tabacum) at the ER-chloroplast contact sites (Tan et al., 2011), also known as PLAMs. BnCLIP1 enzyme exhibited a discrete localization on the outer envelope membrane at the junction between the ER and plastids. The subcellular distribution of a protein such as BnCLIP1 at the PLAM is consistent with a potential role of these sites in the coordinated synthesis of lipids between the ER and plastids (Tan et al., 2011). Yet, it is unknown whether proteins of this kind have only a biosynthetic role at the PLAMs, such as lipid synthesis and transport, or have a scaffolding role to tether the two organelles together. Using an optical trapping and tweezer system, physical contacts of the ER with the Golgi also have been established. In plant cells, the Golgi apparatus is dispersed into polarized mini-stacks that are motile (Boevink et al., 1998; Nebenführ et al., 1999). By trapping and pulling Golgi stacks in cells coexpressing fluorescent reporters for the ER and for the Golgi, it was shown that the pulling of Golgi stacks in cells where the movement of the ER and the Golgi was chemically inhibited was followed by a movement of an ER tubule in association with the Golgi (Sparkes et al., 2009b). Recently, using a similar approach, it was shown that overexpression of a truncated membrane-anchored Golgi matrix protein AtCASP lacking the coiled-coil domain in the cytosolic region could weaken the ER-Golgi connections, supporting the presence of proteinaceous scaffolding that tethers the ER and the Golgi together (Osterrieder et al., 2017). Although the identification of AtCASP as a putative ER-Golgi tether is a landmark in plant cell biology, it will be important to pursue further the identification of the proteins responsible for the tethering of different membranes with the plant ER not only at the Golgi but also with the PM and the other organelles in the plant cell. This is a field that is considerably lagging behind compared with nonplant cell systems (Rocha et al., 2009; Eden et al., 2010; Kornmann et al., 2011; Stefan et al., 2011; Michel and Kornmann, 2012; Murley et al., 2015). One of the most noticeable features visible through microscopy analyses of live cells expressing bioreporters of the ER is the high dynamicity of this organelle; indeed, the ER tubules and cisternae continually move and rearrange, evolving the overall architecture during time and cell development. Curiously, in plant cells, the ER does not have only one form. Indeed, during the life cycle of a plant cell, the ER undergoes considerable reorganization of the morphology and dynamics (Fig. 1; Ridge et al., 1999; Stefano et al., 2014; McFarlane et al., 2017). Initial reports that the ER assumes different shapes were provided more than 2 decades ago in analyses using a fluorescent probe for lipids (DiOC6) as well as a fluorescent protein targeted to the bulk ER (Hepler et al., 1990; Ridge et al., 1999). The DiOC6 dye was used to explore the structure of the ER in moss during bud formation. Noticeable changes in ER architecture were established, starting from a dense meshwork of membranes that was reorganized into an open reticular network as the cell underwent growth. Through analyses performed using a fluorescent protein tagged to the ER on root and hypocotyl cells of Arabidopsis (Arabidopsis thaliana), it was possible to distinguish an ER characterized by lamellar sheets in early phases of cell expansion followed by a change toward a reticulate tubular structure, which is typical of the ER of fully expanded cells (Ridge et al., 1999). It also was noticed that in fully expanded root epidermal cells that give origin to root hairs, the reticular ER network was condensed at the sites where the root hairs are formed (Ridge et al., 1999). The organization of the ER morphology becomes even more puzzling in dividing Pinus root cells in which the ER at preprophase and prophase is spatially rearranged to overlay the MTs (Quader and Zachariadis, 2006). Indeed, treatment with oryzalin, a MT inhibitor, affects the formation of the tubular ER (tER)-preprophase band, tER-metaphase spindle, and tER phragmoplast, suggesting that at least in Pinus root cells the ER network organization may depend on MTs during mitosis and cytokinesis. Although together these results support that the ER assumes different morphology during cell growth, it is yet to be shown whether there may be a functional link between the shape of the ER and the ER function at specific stages of cell growth and development. It has been shown recently that mutations in the ER-shaping protein Root Hair Defective3 (RHD3) compromise not only the overall organization of the ER and the transition from extensive sheet-like form to a reticulated pattern, but also cell elongation (Stefano et al., 2014). These results therefore support the existence of a close connection between the ER shape and cell elongation, although the underlying mechanisms have yet to be defined. Based on the evidence that the loss of RHD3 alters the distribution of auxin in roots (Stefano et al., 2015), increases the cell’s phospholipid content, and leads to an attenuation of the UPR of the ER (Lai et al., 2014; Maneta-Peyret et al., 2014), the relationship between ER morphology and cell growth may be the sum of several processes that are affected by the alteration of ER architecture. In plant cells, ER architecture is correlated to cell expansion. Shown are confocal images of wild-type Arabidopsis Col-0 cotyledon epidermal cells expressing an ER lumen marker ERYK (Nelson et al., 2007) at different phases of cell expansion. Note the change in the morphology of the ER network, which from a most cisternal appearance in cells 3 d after germination (DAG) assumes progressively a more reticulated organization as cells expand. Scale bar = 5 μm. In fully expanded cells, the ER network is highly pleiotropic. Despite an anchoring to stable EPCSs, the network undergoes profound rearrangements through tubule interconversion into other tubules as well as fusion of tubules with cisternae. While processes of fission/fragmentation have been verified in nonplant cells systems, such as sea urchin, starfish, lacrimal cells, and nonneuronal cell lines (Terasaki and Jaffe, 1991; Jaffe and Terasaki, 1993; Subramanian and Meyer, 1997; Dayel et al., 1999; Ribeiro et al., 2000; Harmer et al., 2002; Kucharz et al., 2009), the common profiles for rearrangement of the plant ER are homotypic membrane fusion and tubule emergence from other tubules. These rearrangements are guided primarily by the cytoskeleton (Sparkes et al., with the MTs a yet MTs ER tubule extension with an compared with and MTs to provide anchoring for the formation of et al., 2014). While the of the proteins the plant ER to the MTs are yet recently two proteins have been shown to link ER to The protein to the of plant-specific et al., 2000). These named and a high of et al., 2000; et al., 2016). and a domain, a putative transmembrane domain, and a cytosolic which an actin-binding et al., 2016). is primarily localized to the ER. In conditions of overexpression of the architecture of the ER changes with a of the cisternal profiles and a network that the a loss of of the ER, reduces its and cell elongation et al., 2016). These results that is an is only one of the components of the other proteins similar functions that may be important in different developmental The other protein in connection is which is a plant-specific protein to a et al., 2014). proteins two important In the C-terminal a coiled-coil domain may be important for protein-protein interactions, and the important domain that this is the actin-binding domain, which may as an to link membranes to the In has been shown to in with the ER. the protein with a fluorescent marker is to conditions of the architecture of the ER into resembling the cytoskeleton (Wang and 2017). Although from a direct interaction of with has not been these results that may function as a between the ER and of a not defects compared with the (Wang and 2017), suggesting that there may be functional the While the cytoskeleton a dynamic for the overall ER ER proteins such as and RHD3 have an important role in the of the are membrane proteins that assume a with their transmembrane regions et al., 2006; et al., Sparkes et al., By into the the form and high of the ER which results in the formation and of tubules et al., et al., 2011). with this overexpression of Arabidopsis a that at the ER tubules and the of ER of the ER lumen and reduces the of lumen in the ER et al., The function of on a conserved domain at the C-terminal region that a putative et al., 2016). of not formation but the function of in et al., 2016). These results are important as support that the function of may not be to their ability to but to the presence of the The plant of et al., Sparkes et al., the of this and the that may of the ER network in has been it be to out with lacking the to the molecular role of this domain in the of ER In addition to the RHD3 has important at the ER. RHD3 is an ER protein with two putative transmembrane A of RHD3 was in a for root defects and of function of RHD3 leads to a elongation of cells of the root (Stefano et al., At a subcellular level, the loss of RHD3 also reduces the formation of to the of the protein to membranes in a (Ueda et al., 2016). In this RHD3 functions to similar such as metazoan and yeast et al., 2011; et al., et al., 2015). it has been shown that the functional of RHD3 may depend on In overexpression of the C-terminal region of RHD3 the ER network overexpression of the region does not (Stefano and Brandizzi, 2014). the C-terminal domain of RHD3 is and it has been shown that treatment of RHD3 of this which in may its ER-shaping function (Ueda et al., 2016). One puzzling RHD3 in relation to ER shape in general the physiological role of ER membrane RHD3 to a of proteins of and The evidence that a double of RHD3 and is and that the loss of RHD3 and 2 et al., suggests that the formation of the tubular ER network is important for the cell. the evidence that in cells the ER does not have a reticulated form the on how the shape of the ER may the of cells at stages of growth compared with In addition to and RHD3 there may be other proteins in ER in nonplant cells, the proteins as ER in with the In have been in the of proteins and One with a fluorescent is localized at the ER with RHD3 et al., 2011). A functional of proteins in the of ER shape is and it be that additional proteins may be in plant ER architecture. In and it has been shown that a domain is for ER In has been in to the most by the ER et al., 2015; et al., 2016). A functional ortholog of has yet to be in and it be yet that other proteins may have functions to or that the of the plant ER on different mechanisms compared with nonplant cell systems. While in cells the rearrangement of the ER network as well as transport of largely depend on in plant cells this role is primarily by and the plant-specific and et al., et al., 2010; et al., Indeed, the loss of the movement of ER, Golgi and et al., et al., which is streaming and Stefano et al., 2014). The role of streaming has not been at a molecular level, but it is to to the presence of a central vacuole that can up to of the cell the movement of the may the of as well as between sites in the cells. The ER network is a organelle that contacts membranes (Andersson et al., et al., Stefano et al., 2015), and it therefore may have a on their Indeed, as plant cells with the changes of the ER architecture from cisternal to tubular the velocity of ER streaming as well as overall streaming increases (Stefano et al., 2014). Based on this evidence and the that the loss of RHD3 the spatial distribution as well as the streaming not only of the ER but also of other organelles, such as the Golgi, mitochondria, and during cell it has been that the ER movement may contribute to the general streaming through the physical connections that it with membranes (Stefano et al., 2014, 2015). a of has been verified in connection with the loss of ER streaming and of ER morphology in an RHD3 (Stefano et al., 2015). These results support the that the dynamic of organelles such as is important to their function and maintain the overall cell are an exciting for the understanding of the plant ER. Although in recent advances have been toward the understanding of how the structure and dynamic architecture of the ER are it is yet unknown how other fundamental of the ER are established, including how ER subdomains and maintain their and their cellular role may ER subdomains have been and the existence of an association between the ER and and the of the molecular in the association of the ER with the PM or have been established. The proteins and processes underlying homotypic tubule fusion also are emerging (Fig. One of the for will be to of the architecture of the ER in relation to other organelles and the cytoskeleton. This will be with electron which structures at a high et al., 2006). of the of this is provided by a recent using at molecular the architecture of EPCSs in nonplant cells et al., 2015). This is to lead to more on the recently of the ER with the cytoskeleton and other components of the endomembrane in plant cells. the association between the ER and other organelles in a plant cell. not in the cell Golgi regions the or ER rearrangement in plant cells.
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