Accumulation of beta-Fructosidase in the Cell Walls of Tomato Roots following Infection by a Fungal Wilt Pathogen

Active defense in plants is associated with marked metabolic alterations, but little is known about the exact role of the reported changes in specific activity of several enzymes in infected plant tissues. β-Fructosidase (invertase), the enzyme that converts sucrose into glucose and fructose, increases upon infection by fungi and bacteria. To understand the relationship between fungal growth and β-fructosidase accumulation, we used an antiserum raised against a purified deglycosylated carrot cell wall β-fructosidase to study by immunogold labeling the spatial and temporal distribution of the enzyme in susceptible and resistant tomato (Lycopersicon esculentum) root tissues infected with the necrotrophic fungus, Fusarium oxysporum f. sp. racidis-lycopersici. In susceptible plants, the enzyme started to accumulate in host cell walls about 72 hours after inoculation. Accumulation occurred only in colonized cells and was mainly restricted to areas where the walls of both partners contacted each other. In resistant plants, accumulation of β-fructosidase was noticeable as soon as 48 hours after inoculation and appeared to reach an optimum by 72 hours after inoculation. Increase in wall-bound β-fructosidase was not restricted to infected cells but occurred also, to a large extent, in tissues that remained uncolonized during the infection process. The enzyme also accumulated in wall appositions (papillae) and intercellular spaces. This pattern of enzyme distribution suggests that induction of β-fructosidase upon fungal infection is part of the plant's defense response. The possible physiological role(s) of this enzyme in infected tomato plants is discussed in relation to the high demand in energy and carbon sources during pathogenesis.     

Evolution of the MIP family of integral membrane transport proteins

Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment. Phylogenetic‘trees’interrelating these proteins and segments are presented.     

A short domain of the plant vacuolar protein phytohemagglutinin targets invertase to the yeast vacuole.

Phytohemagglutinin (PHA), the seed lectin of the common bean, accumulates in protein storage vacuoles of storage parenchyma cells in cotyledons. When expressed in yeast, PHA is efficiently targeted to the yeast vacuole [Tague and Chrispeels (1987). J. Cell Biol. 105, 1971-1979]. To identify vacuolar sorting information in PHA, a series of 3' deletions of the PHA gene were fused in-frame to a truncated yeast invertase gene. An amino-terminal portion of PHA composed of a 20-residue signal sequence and 43 residues of the mature protein efficiently targeted invertase to the yeast vacuole. Internal deletions in a short PHA-invertase fusion showed that targeting information exists between residues 14 and 23 of mature PHA. Based on examinations of three-dimensional structures of related lectins, only a portion of these residues would be available on the surface of PHA for interaction with a putative receptor. Amino acid replacements at these positions in a PHA-invertase hybrid caused secretion of the invertase. The results indicate the presence of a vacuolar targeting domain in PHA that is centered at position 19 of the mature protein. This sequence of PHA also shows sequence identity to a vacuolar sorting domain characterized in yeast carboxypeptidase Y. Single amino acid alterations in a short PHA-invertase hybrid protein that caused the highest levels of secretion introduced a glycosylation site at position 21 of PHA. This observation suggests that glycan addition may interfere with recognition of a sorting determinant. These same amino acid changes did not dramatically increase secretion in a long PHA-invertase fusion or in PHA itself. Thus, a second domain of PHA may function in concert with the first one to bring about correct targeting of PHA.     

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.Abbreviations IgG immunoglobulin G - M_r relative molecular weight - PBS phosphate-buffered saline - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Synthesis of an integral protein of the protein-body membrane in Phaseolus vulgaris cotyledons

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm^-3. Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with ³H-amino acids for 2–3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999–1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425–1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.Abbreviations EDTA ethylenediamino-tetraacetic acid - ER endoplasmic reticulum - IMP integral membrane protein - PB protein body - PBM protein-body membrane - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A precursor of the reserve-protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons

Developing cotyledons of Phaseolus vulgaris L. were labeled for 30 min with [³H] amino acids, homogenized, and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [³H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm^-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl₂ indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER, and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [¹⁴C] amino acids and [³H] glucosamine or with [¹⁴C] amino acids and [³H] mannose, the [³H]/[¹⁴C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility on SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.