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.

Living with genome instability: plant responses to telomere dysfunction.

Loss of telomere function in metazoans results in catastrophic damage to the genome, cell cycle arrest, and apoptosis. Here we show that the mustard weed Arabidopsis thaliana can survive up to 10 generations without telomerase. The last five generations of telomerase-deficient plants endured increasing levels of cytogenetic damage, which was correlated with developmental anomalies in both vegetative and reproductive organs. Mutants ultimately arrested at a terminal vegetative state harboring shoot meristems that were grossly enlarged, disorganized, and in some cases, dedifferentiated into a callusoid mass. Unexpectedly, late-generation mutants had an extended life-span and remained metabolically active. The differences in plant and animal responses to dysfunctional telomeres may reflect the more plastic nature of plant development and genome organization.

Purification and characterization of a secreted purple phosphatase from soybean suspension cultures.

We purified and partially sequenced a purple (lambda(max) = 556 nanometers) acid phosphatase (APase; EC 3.1.3.2) secreted by soybean (Glycine max) suspension-culture cells. The enzyme is a metalloprotein with a Mn(2+) cofactor. This APase appears to be a glycoprotein with a monomer subunit molecular weight of 58,000 and an active dimer molecular weight of approximately 130,000. The protein has an isoelectric point of about 5.0 and a broad pH optimum centered near 5.5. The purified enzyme, assayed with p-nitrophenyl phosphate as the substrate, has a specific activity of 512 units per milligram protein and a K(m) of approximately 0.3 millimolar; phosphate is a competitive inhibitor with a K(i) of 0.7 millimolar. This APase is similar to one found in soybean seed meal but dissimilar to that found in soybean seedlings.

Comparisons of Golgi structure and dynamics in plant and animal cells.

The Golgi apparatus of both higher plant and animal cells sorts and packages macromolecules which are in transit to and from the cell surface and to the lysosome (vacuole). It is also the site of oligosaccharide and polysaccharide synthesis and modification. The underlying similarity of function of plant and animal Golgi is reflected in similar morphological features, such as cisternal stacking. There are, however, several fundamental differences between the Golgi of plant and animal cells, reflecting, in large part, the fact that the extracellular matrices and lysosomal systems differ between these kingdoms. These include 1) the form and replication of the Golgi during cell division; 2) the disposition of the Golgi in the interphase cell; 3) the nature of "anchoring" the Golgi in the cytoplasm; 4) the genesis, extent, and nature of membranes at the trans side of the stack; 5) targeting signals to the lysosome (vacuole); and 6) physiological regulation of secretion events (constitutive vs. regulated secretion). The degree of participation of the Golgi in endocytosis and membrane recycling is becoming clear for animal cells, but has yet to be explored in detail for plant cells.

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

Helianthinin 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 beta-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.

Convergence of the endocytic and lysosomal pathways in soybean protoplasts.

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.

Expression, glycosylation and secretion of phaseolin in a baculovirus system.

In this report, we describe the efficient expression and glycosylation, in insect cells, of ß-phaseolin polypeptides (M r 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 (M r 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/10(6) 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 (G¼C) 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.

Cell-fractionation analysis of glucan synthase I and II distribution and polysaccharide secretion in soybean protoplasts : Evidence for the involvement of coated vesicles in wall biogenesis.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [(3)H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [(3)H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.

Isoelectric focusing of plant cell protoplasts: separation of different protoplast types.

The surface charge of plant protoplasts has been measured by a new technique, isoelectric focusing. The protoplasts were loaded in a dextran density gradient over which a pH gradient was superimposed. When voltage was applied, protoplasts moved to a point in the gradient corresponding to their isoelectric point (pI). The pI of the protoplasts varied with the compounds used for pH gradient generation. Using commercial ampholytes for pH gradient formation, the pI of all protoplasts tested was 4.4 +/- 0.2, and viability following electrophoresis was low. Using an acetate/acetic acid mixture to generate the pH gradient, the pI of protoplasts varied from 3.7 to 5.3 depending on the species and tissue type of the parental cells. Postelectrophoresis viability was high. Using isoelectric focusing techniques, it was possible to separate mixtures of protoplasts derived from different species of plants.

Cytological and biochemical requirements for the establishment of a polar cell.

Our research is aimed at understanding the biochemical and cytological basis of cell polarity in zygotes of the brown alga, Fucus distichus L. Powell. One manifestation of this polar cell is the localization of a sulphated fucan polysaccharide (F2) in only one region of the zygote cell surface, the rhizoid cell wall. The focus of this paper is centered around the mechanism responsible for the directional transport of Golgi vesicles containing F2 and the biochemical properties of F2 that might specify its localized fate. Recent findings indicate that the various sulphated polysaccharides in the brown algae are complexes resulting from linkages of two basic polymers: an alpha-(1----2)-linked fucan that contains high levels of ester sulphate (F3), and a uronic acid-rich polymer (F1). The fucan complex F2, which is localized in the rhizoid wall, is composed of a fucan sulphate core (F3) to which uronic acid polymers (similar to F1) are attached. Our results, using a purified endoguluronate lyase, indicate that guluronate bridges link these subunits of F2. The carbon backbone of F2 is not synthesized de novo after fertilization. However, F2 is sulphated, and possibly assembled, beginning 10 h after fertilization, after which it is locally inserted into the rhizoid wall, and held in the wall structure only by calcium ionic bonds. Although sulphation is required for localization of F2, it is not known if the uronic acid side-chains are also assembled at the time of sulphation, and/or required for localization. The fact that F3 (F2 without the side-chains) is secreted uniformly into the zygote wall suggests that the uronic acid chains of F2 may play a critical role in its localization. A sulphated F2 alone is not sufficient for its localization since in the presence of cytochalasin, vesicles containing F2 are not transported to the rhizoid. Recent studies point to a central role for a cytoskeletal element, possibly microfilaments, in the directional transport of these vesicles. We have used the techniques of isoelectric focusing and electrophoretic mobility to study surface charge of these Golgi vesicles to determine if charge might be one factor that specifies their localization. Vesicles that contain the sulphated fucan F3 are secreted randomly and have the same surface charge as those containing F2 that are directionally transported. However, there is no stable endogenous electrical current at the time when F3 vesicles are randomly secreted, whereas a current is detectable when F2 vesicles are localized.(ABSTRACT TRUNCATED AT 400 WORDS)

The isolation of coated vesicles from protoplasts of soybean.

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant "clathrin", had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.

Isoelectric focusing of plant cell membranes.

An isoelectric focusing method is described that discriminates plant cell organelle populations on the basis of surface charge. The isoelectric points (pI values) of the Golgi apparatus, the mitochondria, and putative plasma membrane from etiolated pea stem cells are reported. The pI of the pea Golgi apparatus is similar to that of the (35)SO(4)-labeled membranes of developing Fucus embryos. The pI of Fucus(35)SO(4)-labeled membranes depends on whether the membranes are being polarly transported to the growing tip or randomly transported to the entire periphery of the zygote. Those subject to polar tip transport have a significantly greater negative surface charge than those being randomly transported. The implication of this result toward an understanding of the localization mechanism is discussed. The method is also capable of subfractionating glucan synthetase II-containing membranes (putative plasma membrane) from peas. The pI of putative plasma membrane from a pea stem homogenate is similar to the pI of the plasma membrane of whole protoplasts of Catharanthus leaf cells. Isoelectric focusing appears to be a useful technique to discriminate membranes and, hence, to provide new information and approaches to study cellular and developmental phenomena.

Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts.

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.