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 network of the ER (endoplasmic reticulum) is set up by cytoskeletal control of the movement and remodelling of polygonal rings of tubules, bundles of tubules and cisternal regions. We have developed a new image analysis tool, persistency mapping, to understand the framework upon which the plant ER remodels. With this new tool, we have explored the network nodes, called anchor/growth sites, that may anchor the network by attachment to the plasma membrane. We have determined how the polygonal ring structure depends on myosin XI-K for ‘opening’ and ‘closing’. With latrunculin B treatment, we have investigated the involvement of actin in the elongation and persistency of the tubules. We also show how the cytoskeleton is involved in directional diffusion within the membrane. This observation may lead to an answer to the question of what function this network structure serves in the cell. We propose that the ER acts as a trafficking network, delivering lipid, protein, calcium and signalling molecules to different regions of the cell. It does so by directional reduced dimensional diffusion. The ER network of tubules restricts the dimensionality of diffusion to near one-dimensional, whereas the cisternae reduce it to near two-dimensional. The cytoskeleton does not drive the diffusion, but participates by providing directionality to the diffusion.

On 27 March 1689, Richard Waller, Fellow and Secretary of the Royal Society presented his “Tables of the English Herbs reduced to such an order, as to find the name of them by their external figures and shapes” to his assembled colleagues at a meeting of the Royal Society. These tables were developed for the novice by being color images, composed in pencil and watercolor, of selected plants and their distinguishing characteristics. The botanical watercolors for the tables are now a Turning‐the‐Pages document online on the website of the Royal Society. However, for the past 320 years, the scientific context for the creation of these outstanding botanical watercolors has remained obscure. These tables were developed by Waller as an image‐based dichotomous key, pre‐dating by almost 100 years the text‐based dichotomous keys in the first edition of Flora Française (1778) by Jean Baptiste Lamarck, who is generally given priority for the development of the dichotomous key. How these large folio images were arranged to illustrate a dichotomous key is unknown, but an arrangement based on Waller's description is illustrated here as leaf‐ordering for the separate hierarchical clusters (tables). Although only 24 species of watercolored dicot herbs out of a total of 65 in the set of watercolors (the others being monocots) are used in these tables, they are a “proof of concept”, serving as models upon which a method is based, that of using a key composed of dichotomous choices for aiding identification.

Plasmolysis of hypocotyl cells of transgenic Arabidopsis thaliana and Nicotiana benthamiana diminishes the dynamics of the remodeling of the endoplasmic reticulum (ER) in the central protoplast, namely that withdrawn from the cell wall, and more persistent cisternae are formed, yet little change in the actin network in the protoplast occurs. Also, protein flow within the ER network in the protoplast, as detected with fluorescence recovery after photobleaching (FRAP), is not affected by plasmolysis. After plasmolysis, another network of strictly tubular ER remains attached to the plasma membrane-wall interface and is contained within the Hechtian strands and reticulum. FRAP studies indicate that protein flow within these ER tubules diminishes. Actin is largely absent from the Hechtian reticulum and the ER becomes primarily associated with altered, branched microtubules. The smaller volume of the central protoplast is accompanied by decreased movement rates of tubules, cisternae, and spheroid organelles, but this reduced movement is not readily reversed by the increase in volume that accompanies deplasmolysis.

Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER? In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm (3-6-mW) laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates (boluses) within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45°C, temperature (heat stress). Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.