Lawrence R. Griffing

Movement and Remodeling of the Endoplasmic Reticulum in Nondividing Cells of Tobacco Leaves

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.

Expression, glycosylation and secretion of phaseolin in a baculovirus system

In this report, we describe the efficient expression and glycosylation, in insect cells, of β-phaseolin polypeptides (Mr 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (Mr 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/106 cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.

ER network dynamics are differentially controlled by myosins XI-K, XI-C, XI-E, XI-I, XI-1, and XI-2

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 remodeling 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 remodeling 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 cisternalization rather than tubulation.

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco.

SummaryHelianthinin is the major 11S seed storage protein of sunflower (Helianthus annuus). Like most seed proteins, helianthinin is encoded by a small gene family; two members of this gene family, HaG3-A and HaG3-D, have been isolated and characterized. Tobacco was transformed with a 6 kb fragment of HaG3-A containing the helianthinin coding region flanked by 3.8 kb upstream and 0.4 kb downstream sequence. Expression of helianthinin was developmentally regulated in seeds of transgenic tobacco plants; furthermore, helianthinin polypeptides were proteolytically processed and targeted to the protein bodies of transgenic tobacco. A fragment of HaG3-A from −2376 to +24 was fused to the β-glucuronidase (GUS) reporter gene and transferred to tobacco. GUS expression driven by this helianthinin upstream region was developmentally regulated in seeds. Germinating seedlings of the same transformant exhibited a time-dependent decrease in GUS activity with none detected by 6 days post imbibition (DPI). Histochemical analysis of GUS activity in embryos and 2 to 5 DPI seedlings showed expression restricted to the cotyledons and upper embryonic axis with none detected at the radicle end. No GUS activity was found in cotyledons, hypocotyls, leaves, and roots of 18 day seedlings or in leaves of an 8 week F1 plant. These results indicate that the cis-regulatory elements required for developmental control of the HaG3-A helianthinin gene are located in a 2.4 kb upstream region of this gene. This region was sequenced together with the upstream region of the HaG3-D helianthinin gene.

Plant ER geometry and dynamics: biophysical and cytoskeletal control during growth and biotic response

The endoplasmic reticulum (ER) is an intricate and dynamic network of membrane tubules and cisternae. In plant cells, the ER ‘web’ pervades the cortex and endoplasm and is continuous with adjacent cells as it passes through plasmodesmata. It is therefore the largest membranous organelle in plant cells. It performs essential functions including protein and lipid synthesis, and its morphology and movement are linked to cellular function. An emerging trend is that organelles can no longer be seen as discrete membrane-bound compartments, since they can physically interact and ‘communicate’ with one another. The ER may form a connecting central role in this process. This review tackles our current understanding and quantification of ER dynamics and how these change under a variety of biotic and developmental cues.

Post Golgi apparatus structures and membrane removal in plants

SummaryIn nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.

Immunolocalization of the major latex proteins in developing laticifers of opium poppy (Papaver somniferum)

Summary The most abundant protein species in poppy latex is a group of low molecular weight peptides which have been termed the major latex proteins or MLPs. In the present investigation the subcellular distribution of MLPs in developing poppy laticifers is examined using ultrastructural immunolocalization. In mature laticifers the MLPs are localized in a subpopulation of the latex vesicles. In some laticifers the protein appears to be compartmentalized within individual vesicles. Associated with the mature laticifers are cells which also contain MLPs in a large central, apparently protein-filled, vacuole. These cells are interpreted to be developing laticifers. Based on these observations a new model for laticifer vesiculation is presented in which fragmentation of a large central vacuole of immature laticifer elements gives rise to the highly vesiculated cytoplasm observed in the mature latex vessel.

FRET analysis of transmembrane flipping of FM4–64 in plant cells: is FM4–64 a robust marker for endocytosis?

Although the styryl dye FM4–64 is now used routinely to monitor endocytosis in plants, the argument about its potential to cytoplasmically and non‐endocytically relocate into a selective set of vesicular compartments persists. To address this question, we determined whether fluorescence resonance energy transfer (FRET) could occur between a cytoplasmically expressed, short‐wavelength excitation green fluorescent protein (GFP) and FM4–64 in Nicotiana benthaminana. After exposure to FM4–64, the root hair plasma membrane and internal organelles became labelled. Under these conditions, no FRET with cytoplasmic GFP was seen. However, if the cells were treated with a low concentration of quillajasaponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. Thereby, we demonstrate that under conditions that do not severely compromise cell viability, the FM4–64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP. Under normal conditions, FM4–64 does not significantly enter the cytosolic side of the membrane, but remains at the plasma membrane or trapped in the organelles of the endocytic pathway. Hence, when the structure or permeability of the plasma membrane is unaltered, FM4–64 dye is a robust marker for endocytosis.

Laser stimulation of the chloroplast/endoplasmic reticulum nexus in tobacco transiently produces protein aggregates (boluses) within the endoplasmic reticulum and stimulates local ER remodeling.

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.

Radioimmunoassay of Morphinan Alkaloids in Papaver somniferum Hypocotyls, Callus, and Suspension Cultures

Summary Hypocotyls of poppy ( Papaver somniferum L. cv. Marianne) were assayed for morphinan alkaloid content using a commercially available radioimmunoassay (RIA) for morphine (Abuscreen, Roche Diagnostics). The hypocotyls showed a sharp rise in morphinan alkaloid content on day 3 following sowing, reachin a plateau (9 ng·mg- 1 fresh wt) between day 4 and day 5. Hypocotyl explants were cultured for up to 2 months on solid nutrient medium containing α-naphthaleneacetic acid. During this time and while hypocotyls formed callus, the morphinan alkaloid content fell to a «maintenance» level of 0.4 ng·mg −1 fresh weight in culture. Callus masses retained laticifer-like cells. If cell suspensions were plated with medium lacking growth regulators, plantlets were regenerated and the level of alkaloids found for hypocotyls was re-established. If plated cell suspensions were cultured in the presence of growth regulators, organs formed, but plantlets did not regenerate and the alkaloid content remained at or near the maintenance level. Over a period of a year callus subcultures lost their laticifer-like cells, their embryogenic potential, and their ability to maintain low-level alkaloid production.