Increase of Free Space Solutes in Tobacco Leaves in Relation to the Localized Cellular Response Following Injections of a Bacterial Protein-Lipopolysaccharide Complex*

Variations in pH, cell ultrastructure, conductivity, calcium ion molarity, reducing sugars, uronic acids and in protein of the intercellular washing fluid were studied at 0, 12, 24 and 48 h in tobacco leaf halves intercellularly injected with a bacterial pr-LPS complex (250 μg-ml⁻¹). These injections induced a localized cellular reaction (LCR) in points opposite to intercellular structured deposits on the plant cell walls. The intercellular fluid pH was higher at 24 h than in the control, but not at 12 and 48 h. The conductivity, the calcium ion molarity, the free and hydrolyzed sugar content were higher at 12, 24 and 48 h than in the control tissue; the uronic acid content was higher at 24 and 48 h, but not at 12 h. There was a peak for, all 5 parameters at around 24 h, when LCR showed its highest activity. The protein content was significantly higher at 24 and 48 h than in the control intercellular fluid. The increase in conductivity, calcium ions, sugar, uronic acids and proteins in the intercellular fluids of the pr-LPS injected tissue were interpreted as direct or indirect effects of the LCR, i.e. as an exocytosis induced by pr-LPS injections. The associated high sugar and uronic acid content of the intercellular fluid at 12 and 24 h was not correlated to its capacity to prevent the hypersensitive confluent necrosis when injected intercellularly into tobacco tissue.     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 4. Scanning Electron Microscope Studies on fractured Leaf Tissue

Leaves of tobacco infiltrated with Pseudomonas pisi were fractured at various times during the course of the hypersensitive reaction to expose cell surfaces within the tissue and mesophyll cell contents. Scanning electron microscopy of cross-fractured mesophyll cells did not reveal any gross change in internal structure during the reaction induction period (0—2 h), but breakdown of tonoplast and collapse of chloroplasts commenced at about 5 h, during the latent period. Death of the mesophyll cells was followed by condensation of cell contents, and pronounced stretching of cell walls, due to desiccation and shrinkage.Between 0—6 h after infiltration, bacteria were largely confined to cell junctions, frequently within droplets. With collapse of the host cells and release of cell fluid, numbers of bacteria increased considerably (many dividing cells), and there was a shift of bacterial distribution to the whole mesophyll cell surface. The progressive desiccation that occurred between 10—20 h prevented further bacterial increase, but numbers of bacteria remained stable. Death of bacteria commenced at about 15 h, and was accompanied by the formation of numerous surface protrusions, which detached and deposited over the whole mesophyll surface.     

Multiplication of Pseudomonas syringae pv. phaseolicola"in planta"

In the compatible combination of the halo blight disease of bean Pseudomonas phaseolicola was able to colonize large areas of the intercellular space of leaves, such that these confluent water congested areas became visible as water-soaked spots. Most of the plant cell walls in the infected region maintained their normal shape, even when the cytoplasm had collapsed. Some inward bending of plant cell walls preceded their rather slow degradation and final replacement by bacterial masses. Neighbouring plant cells appeared to be metabolically active. In resistant leaves no indications of active bacterial attachment or encapsulation could be observed. However, bacteria appeared to be more densely packed in resistant leaves, and relatively more plant cells completely collapsed as compared with susceptible leaves. From 8—14 days after inoculation, the bacterial concentration did not change much in susceptible or resistant leaves, indicating the absence of bactericidal components. Even Pseudomonas pisi snowed some multiplication in bean leaves (immune reaction), but its growth stopped earlier than that of P. phaseolicola. in the resistant cultivars, probably due to a different mechanism of resistance. Although less bacteria were determined in the intercellular washing fluid (IF) compared with leaf homogenates, the high bacterial concentrations in the IF supported our observation that an effective encapsulation of bacteria in resistant leaves did not occur.     

Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress

Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.     

Histochemistry of Tobacco Mesophyll Cell Walls Pretreated with Bacterial Protein-lipopolysaccharides

Prevention of hypersensitive confluent-necrosis in tobacco mesophyll, caused by Pseudomonas syringae pv. aptata, and tolerance towards the incompatible bacterium are induced 48 h after intercellular injection of protein-lipopolysaccharide complexes. Histochemical analysis and ultrastructural observations were carried out to determine whether there was an association between this tolerance state and cell wall alterations due to callose, lignin and/or suberin deposition either before or after challenge with the incompatible bacterium. No evidence was obtained for such wall alterations.     

Effect of Salicylic Acid and Phenylserine on the Hypersensitive Reaction of Tobacco to Tobacco Mosaic Virus

Injection of leaves of tobacco (Nicotiana tahacum cv. ‘Xanthi’ nc) with salicylic acid (SA) or phenylsene (PS) had an effect on the local lesion development caused by tobacco mosaic virus (TMV), depending upon the concentration used and the time interval between injection and challenge inoculation. Maximum reduction in lesion size was obtained with 0.75 mM SA or with 8 mM PS. Concentrations higher than 1 mM SA or 25 mM PS damaged the leaf tissue, PS being far less toxic than SA. The leaves responded rapidly to injection with SA or PS. A time interval of only 1 h between injection and TMV inoculation reduced the lesion size significantly. Isolated tobacco cell walls incubated with SA yielded carbohydrate fractions capable of reducing lesion size significantly after injection. Cell walls incubated without SA or with PS did not yield active carbohydrate fractions.     

An Electron Microscopic Study of the Infection of Spikelets of Rice by Pyricularia oryzae

Penetration and colonization of spikelet tissue of a susceptible line of rice. ZTS, by Pyricularia aryzae was investigated by scanning and transmission electron microscopy. Application of a modified embodding procedure in which the period of resin infiltration was extended, gave excellent fixation of the hard spikelet tissue. Conidia germinated to produce appressoria and the epidermal cell walls were penetrated within 24 h. Hyphae had colonized sclerenchyma, parenchyma, vascular bundles, andlarge trichomes within 48 h after inoculation. Invasion of the sclerenchyma appeared to occur through pit-pairs, where the host cell wall is thin. Conidiophores erupted directly from the epidermis, from minute protuberances and from large trichomes on the spikelet 4 days after inoculation.     

Transmission Electron Microscopy of Susceptible and Resistant Tomato Leaves Following Infection with Pseudomonas syringae pv. tomato

Ultrastructural changes in tomato leaves of susceptible cv. Peto 95 and resistant cv. Ontario 7710 infected with Pseudomonas syringae pv. tomato were followed by transmission electron microscopy. Up to 48 hours from the inoculation host cells of both cultivars looked quite normal and no bacteria were visible in the intercellular spaces; bacterial cells were found only in the substomatal chambers. Afterwards, the leaf cells of cv. Peto 95 began to degenerate and bacteria invaded the intercellular spaces which seemed enlarged. After 15 days the disorganization was complete: tomato cells were plasmolyzed and the intercellular spaces were filled with bacteria. In the leaves of resistant cv. Ontario 7710 no bacteria were observed later than 48 hours and no visible modifications occurred up to 15 days after the inoculation.     

Ultrastructure in Bean Leaves Infiltrated with Bacterial Extracellular Polysaccharides

Ultrastructural observations were carried out in bean leaf tissue infiltrated with bacterial extracellular polysaccharides. These investigations revealed changes m the cell fine structure, especially related with chloroplast organization. Up to 24 h after EPS infiltration alterations consisted in irregularly running thylakoids and distortions of stacked regions. Invaginations of the plastid envelope and appearance of cytoplasm pockets inside the stroma were also noted. The most severe alterations, consisting in stroma dilations and envelope infoldings, were noted 24 h after EPS infiltration, parallehng the yellowing of the treated leaf areas; chloroplast shrinkage and collapse of thylakoid system were also sometimes observed. Chloroplast ultrastructure generally recovered 48 h after treatment; at this time local detachment of the plasma membrane and vesicle formation in the periplasmic space were observed, resembling a non specific, locahzed cellular response. The observed, permanent chlorosis and ultrastructural alterations suggest an interference of the EPS in the global metabolism of the bean mesophyll cell.     

Relationship of Footpad Sensitivity to Purified Protein Derivatives and Resistance to Airborne Infection with Mycobacterium tuberculosis of Mice Vaccinated with Mycobacterial Cell Walls

Footpads of mice sensitized by oil-treated Bacillus Calmette-Guérin (BCG) cell walls given either intravenously, subcutaneously, intradermally, intraperitoneally, or intramuscularly became swollen and reddened after injection of purified protein derivative (PPD). This reaction, greatest after intradermal and subcutaneous sensitization, generally reached a maximum about 24 hr after challenge and was still marked at 48 hr. The histological response was characterized by infiltration with both polymorphonuclear and mononuclear cells. The proportion of mononuclear cells increased with time and they predominated at 48 hr. The footpad reaction could be detected as early as 1 week after sensitization and persisted for at least 37 weeks. Footpad sensitivity to PPD and acquired resistance to airborne infection with Mycobacterium tuberculosis H37Rv were correlated in that (i) both reached a peak approximately 1 month after sensitization of the mouse, and (ii) cell walls treated with NaOH or given without oil neither protected mice against challenge infection nor sensitized them to PPD. Although, as we reported previously, mice vaccinated subcutaneously or intradermally exhibited little or no enhanced resistance to experimental infection, mice given oil-treated cell walls by these routes were highly sensitive to footpad inoculation of PPD. Therefore, the footpad test cannot be used to determine immunity of the mouse to pulmonary infection with tubercle bacilli.     

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 5. Inhibition of RNA Synthesis in Mesophyll Cells

The synthesis of mesophyll cell RNA in tobacco leaf tissue infiltrated with the incompatible bacterium Pseudomonas pisi was investigated by light and electron microscope autoradiography of H³-uridine uptake. Interpretation of the light microscope quantitative data was complicated by the oscillation in levels of cytoplasmic RNA synthesis that resulted from the physical process of fluid infiltration (seen in the control tissue). Direct comparison with the control showed that the presence of bacteria resulted in a rapid decrease in the synthesis of host cell RNA. This effect was clear within the first 1½h (induction period) of the hypersensitive reaction, where it was particularly marked in the cytoplasm of palisade cells. Electron microscope autoradiography showed that the presence of bacteria did not cause complete cessation of RNA synthesis in mitochondria and chloroplasts. The early inhibition of RNA synthesis preceeded fine structural (degenerative) change, and should be regarded as one of the primary events associated with the induction of host cell necrosis.     

The protective role of topical propolis on experimental keratitis via nitric oxide levels in rabbits

The aim of this study was to investigate antioxidant, anti-inflammatory, and antibacterial properties of propolis in the treatment of experimental Staphylococcus aureus keratitis. Twenty young New Zealand white rabbits were used in this experiment. Staphylococcus aureus were given by intrastromal injection to 16 rabbits and 4 rabbits were used as control group (Group 1). Group 2 was treated with phosphate-buffered solution drops; Group 3 was administered ethanolic extract of propolis drops; Group 4 received topical ciprofloxacin drops; Group 5 was treated with topical ciprofloxacin drops along with ethanolic extract of propolis drops. The eyes were examined by slit lamp to assess corneal opacity. And then, corneas were removed to determine nitric oxide (NO) levels and count bacteria. Corneas were also evaluated histologically. Corneal NO concentration in gruop 5, treated with a combination of propolis and ciprofloxacin was determined significantly lower (10.0± 1.8 μmol/g wet tissue) than in Group 4, treated with ciprofloxacin (24.0± 3.1 μmol/g wet tissue), from Group 3, treated with propolis (15.6± 1.8 μmol/g wet tissue), and treated with PBS (44.7± 7.8 μmol/g wet tissue). There were significantly fewer bacteria in eyes that received propolis plus ciprofloxacin than in eyes treated with ciprofloxacin (p = 0.0001) or propolis (p = 0.0001) or eyes treated with PBS (p = 0.0001). The light microscopic examination revealed that the control group showed normal corneal morphology. In the nontreated group, sections of the stromal infiltration revealed the presence of inflammatory cells, which were diffusely distributed (p < 0.05). Administrations of ciprofloxacin plus propolis resulted in a significantly reduced histological damage with fewer bacterial inoculation of the corneal stroma in comparison with the other groups (p < 0.05). Based on these findings, we suggest that ethanolic extract of propolis has antioxidant, anti-inflammatory, and antibacterial properties for S. aureus keratitis in rabbits.     

Die Lokalisation der Peroxidase-Isoenzymgruppe GI in der Zellwand von Tabak-Geweben

Summary By vacuum infiltration of intercellular spaces of tobacco tissues it is possible to extract substances from cell walls which move freely in the walls. The peroxidases (E.C. 1.11.1.7) contained in these extracts are predominantly isoenzymes of G_I (fast migrating anodic group) as was shown by discelektrophoresis of the extracts. As has been demonstrated previously G_I is not present in the protoplast; therefore G_I is the typical cell wall fraction of tobacco peroxidases. Different tissues of tobacco always differ in the isoenzyme pattern of G_I. This pattern also changes during tissue development. We can therefore say that there exists an enzymatic differentiation of plant cell walls during development. As G_I is not bound to the walls, it always appears in high amounts in crude extracts of plant material. Therefore G_I is always called the soluble cytoplasmic fraction, but our investigations clearly demonstrate that G_I is localized in cell walls only. Beside G_I there are much smaller amounts of G_III (slow migrating cathodic group) and if present in the tissue G_II (slow migrating anodic group) detectable in the infiltration fluids of intracellular spaces. G_III and G_II are localized mainly in the protoplast. But they are also bound to the walls, ionically in the case of G_III and covalently in the case of G_II.

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Abkürzungen MDH Malatdehydrogenase (E.C. 1.1.1.37) - G_I, G_II, G_III Enzymgruppen der Peroxydase - FG Frischgewicht     

Contribution of folate biosynthesis to Ralstonia solanacearum proliferation in intercellular spaces

The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.     

Contribution of Folate Biosynthesis to Ralstonia solanacearum Proliferation in Intercellular Spaces

The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.     

Acquired Resistance in Hypersensitive Tobacco against Tobacco Mosaic Virus, Induced by Plant Cell Wall Components

Isolated cell walls from Nicotiana tabacum L. cv. Xanthi-nc were partially digested with enzymes present in the fungal preparation cellulase Onozuka R-10. Released carbohydrate material was separated from the enzymes by gel filtration and fractions were injected into the intercellular spaces of one half of each of several Xanthi-nc leaves. Two to three days after injection the whole leaves were inoculated with TMV. A large reduction in mean lesion diameter was observed in the injected areas compared with those in untreated tissue.     

Role of intercellular adhesion molecule 1 in indomethacin-induced ileitis

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.     

Relative importance of fungi and bacteria in the decomposition of phragmites leaves

The relative importance of fungi and bacteria in the decomposition ofPhragmites leaves was studied in experiments using antifungal and antibacterial antibiotics. Fungi and bacteria were responsible for almost equal proportions of the respiration of the dead leaves after 35 days exposure in a lake, but fungi respired very little after 122 days. The populations of both fungi and bacteria declined between 35 days and 122 days, but fungi declined more. The amount of weight loss ofPhragmites leaves caused by fungi and bacteria was similar after 35 days. Micro-organisms not affected by antibiotics played a significant role in both respiration and loss in weight of leaves.     

Vegetative anatomy of subtribe Orchidinae (Orchidaceae)

Leaves in Orchidinae are essentially glabrous; anticlinal walls of foliar epidermal cells arc basically straight-sided to curvilinear, and cells arc fundamentally polygonal on both surfaces; adaxial cells are larger than abaxial cells. Stomata arc anomocytic and usually only abaxial and superficial; substomatal chambers are small to moderate; outer and inner guard cell ledges are mostly small. There is no hypodermis nor are there fibre bundles. Mesophyll is homogeneous, chlorcnchyma cells arc thin-walled, and intercellular spaces numerous. Crystalliferous idioblasts abound. Vascular bundles are collateral, organized in a single series. and lack associated sclerenchyma. Bundle sheath cells are thin-walled and chlorophyllous. Stems are glabrous; stomata arc frequent in one species, lacking in others. Cortical cells are oval to circular, thick-walled, and interspersed with triangular intercellular spaces. Ground-tissue cells are circular, and triangular intercellular spaces are present. Vascular bundles arc collateral and scattered throughout the ground-tissue or are absent from the central ground-tissue. Epidermis in absorbing roots is one-layered and non-velamcntous. Exodcrmal cells are thin-walled and dead cell walls bear tenuous scalariform bars; some species lack an exodermis. Outer cortical cells are polygonal and lack intercellular spaces; middle layer cortical cells are rounded with triangular intercellular spaces; inner layer cells are polygonal and lack intercellular spaces. Endodermis and pericycle are thin-walled and one-layered. Vascular cylinder is mostly 7–9-arch with xylcm and phloem components alternating regularly; vascular tissue is embedded in parenchyma; pith cells are parenchymatous, polygonal, thin-walled and lack intercellular spaces. Root tubers generally bear a velamen of variable thickness; bulbous-based unicellular hairs frequently form a dense mat; exodermal cells are thin-walled; dead cells have scalariform bars, passage cells are sparse. Ground-tissue consists of rounded water-storage and assimilatory cells interspersed with triangular or quadrangular intercellular spaces; peripheral cells arc polygonal lacking intercellular spaces. Vascular tissue consists of monarch to pentarch meristeles distributed thoughout the ground-tissue each surrounded by a uniscriale endodermis of thin-walled cells. Thin roots ofPlalanthera exhibit a typical central cylinder surrounded by a homogeneous cortex uninterrupted by meristeles; thicker roots show a central vascular cylinder and cortex in which meristeles are also present; in globoid root tubers there is no central cylinder, and the ground-tissue is replete with scattered meristeles. Because the central vascular cylinder in Platanthera gives rise to branches (meristeles), these represent components of a single vascular system and are not separate stelar entities as implied by the use of the term ‘polystele’.