Common responses of cultured soybean cells to 2,4-D starvation and fungal elicitor treatment

The patterns of in vivo protein synthesis in soybean cell suspensions were compared by polyacrylamide gel electrophoresis after the cells had been submitted to different stress conditions : treatment with Phytophthora megasperma (Pmg) cell wall elicitors, 2,4-D starvation and heat shock (HS) temperatures. Changes in protein synthesis patterns induced after elicitation of cell suspensions or after infection of soybean hypocotyls by Pmg were found to be similar to changes brought about by auxin starvation of the cells. Changes common to both stress situations involve a prominent 17 kDa peptide family and 27, 29, 35 and about 45 kDa peptides. Moreover, defense reactions, i.e. glyceollin accumulation and synthesis of chalcone synthase (CHS) were also strongly stimulated in auxin-starved cells. On the contrary, although characteristic sets of low molecular weight heat shock (HS) proteins were synthesized by cells grown at 37°C, no clear similarity was observed with peptides characteristic of auxin-starved cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Pmg Phytophthora megasperma Drechs f.sp.glycinea - HS heat shock - PR pathogenesis-related - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - IEF isoelectrofocusing - iP isoelectric point - kDa kilodalton - P17 17 kDa peptide group of soybean cells cultured in vitro - CHS chalcone synthase     

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

Promiscuous,non-catalytic,tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>) plants

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.     

Direct observations on generative cells isolated from pollen grains of Haemanthus katherinae baker

Generative cells from mature pollen grains of Haemanthus katherinae Baker (African blood lily) were isolated by means of a simple squash method and observed by differential interference contrast (DIC), fluorescence and polarizing microscopy. The isolated cells appeared structurally similar to those observed in vivo and gave no evidence of a typical cell wall. Their viability was confirmed using the fluorescein diacetate test. The cell shape changed rapidly as the sucrose concentration of the medium was varied. The squash method of isolating generative cells holds promise for the direct and experimental study of these cells, especially in the living state.Abbreviations FDA Fluorescein diacetate - PAS Periodic-acid-Schiff - DIC differential interference contrast - NA numerical aperture     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Elicitation of anthocyanin production in cell cultures of carrot (Daucus carota L) by using elicitors and abiotic stress

Summary A cell line of carrot (Daucus carota L) which produces anthocyanin was subjected to various elicitors and abiotic stresses: The elicitors tested were culture filtrates (CF) and cell extracts (CE) of certain bacteria and yeasts. The abiotic stresses were salts of certain metal ions. The production increase obtained with cell extracts of Bacillus cereus. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were 49, 72, 45 and 41% respectively over the control. Maximum elicitation was obtained with elicitor derived from cell extract of the yeast Rhodotorula rubra where it enhanced anthocyanin production by two fold. The abiotic stress agents Ca, Mn, Zn, Co, Fe & V enhanced anthocyanin production. Of all the metal ions tested Ca was the most effective. The elicitation process was governed by the type and level of elicitor.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Electrical aspects of division control inSaccharomyces

Readily discernable changes in electrical character take place during the division cycle of the yeast,Saccharomyces cerevisiae. By using the technique of cellular spin resonance, the electrical polarizability of individual cells can be seen to change with the life cycle, as determined by cell morphology. The polarizability changes indicate that during the advance through the life cycle, the ionic double layers associated with the outer cell wall gradually tighten.     

Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Localization and release mechanism of cellulases in Trichoderma reesei QM 9414

Summary A significant increase in the extracellular yield of -glucosidase was observed when Trichoderma reesei QM 9414 was cultivated on a cellulose medium containing chitin. Measurement of enzyme activities in the various fractions of the mycelium revealed that endoglucanase was truly extracellular while -glucosidase was cell wall bound. Treatment of Trichoderma mycelium with cell wall degrading enzymes (produced from Trichoderma) led to a release of -glucosidase from the mycelium. Apparently chitin, in the presence of cellulose, induces the synthesis of chitinase and other cell wall lytic enzymes which promote release of the intramural -glucosidase into the medium.     

Stimulation of cell division by membrane-active agents

Agents that decrease membrane stability (e.g. dimethyl sulfoxide, lysolecithin, sodium oleate, and short-chain alcohols) stimulate multinucleoid, serpentine filaments of Agmenellum quadruplicatum strains SN12 and SN29 to divide into cellular equivalents within approximately one generation time. Agents that increase membrane stability (e.g. long-chain alcohols) antagonize this timulation. Thus, the physical properties of the cell membrane appear to be involved in the regulation of cell division. These observations suggest that the invagination of the cell wall may be regulated by agents that interact with the plasma membrane and with enzymes involved in peptidoglycan synthesis.     

Production of reserpine and its optimization in cultured Rauwolfia serpentina Benth. cells

Cultured R. serpentina cells have been maintained on modified Linsmaier-Skoog medium for over 13 years. These cultured cells produced much more ajmaline (0.005–0.012% dW) than reserpine (0–0.003% dW). Selection of callus which survived the stress induced by alteration of the medium composition including hormones, was repeated over several generations. Surviving callus was then transferred back to the original liquid growth medium and subculture continued, during which time the cells exhibited a return to their pre- stress rate of growth, enhanced reserpine production, and a decrease in ajmaline production. R. serpentina cell suspension cultures selected as described and serially subcultured in fresh growth medium every 3 weeks consistently produce reserpine at a yield of approximately 0.03–0.06% dW.Abbreviations LS Linsmaier Skoog(1965)medium - ML Modified Linsmaier Skoog medium - B5 Gamborg(1968)medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA Indole-3-acetic acid - BA Benzyl adenine     

Techniques for enhanced release of leaf protoplasts in Populus

Microscopic examination of Populus leaf tissue following enzyme treatment revealed two factors contributing to low protoplast yields: (1) poor penetration of the enzymes into the tissue, and (2) entrapment of protoplasts in leaf debris during protoplast purification procedures. A simple combination of rapid grinding of the tissue in an Omni-mixer prior to enzyme treatment and forceful washing of leaf-debris after digestion provided high exposure of the cells, uniform digestion, and high yields of protoplasts of two Populus clones. Protoplasts exhibited cell wall regeneration and long-term viability in culture. The relative yield advantages of the techniques varied with the inherent digestibility of each clone but could produce up to 600 percent greater protoplast yields in a woody plant species in which protoplast isolation was previously limited. The techniques were also applicable to an herbaceous species, Solanum etuberosum.Abbreviations BA benzyladenine - NAA naphthalene acetic acid - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - (NC-XXXX) North Central Forest Experiment Station accession number assigned to Populus hybrid clones     

Messenger RNA induction in cellular salt tolerance of Alfalfa (Medicago sativa)

A salt tolerant alfalfaMedicago sativa L. cell line (HG2-N1) has been selected for growth in 171 mM NaCl. The salt tolerance characteristic is stable and is retained after growth in absence of salt selection for two months.In vitro translation was used to compare mRNA composition from the salt tolerant HG2-N1 and parent salt sensitive HG2 cell lines grown in the presence and absence of 171 mM NaCl. The results suggest that the mRNA composition differs between HG2-N1 and HG2 in a number of RNA species. The salt tolerant HG2-N1 shows both increases and decreases in specific polypeptides as compared to HG2. Many of the enhanced polypeptide bands from mRNA in the salt tolerant HG2-N1 variant appear to be constitutively expressed, since they can be detected from HG2-N1 cells grown in presence and absence of NaCl, but the expression of a few bands may depend on the presence of added NaCl. Most enhanced polypeptides, which are detected from mRNA in the salt tolerant variant HG2-N1 (grown on NaCl) are different from polypeptide bands enhanced in the salt sensitive HG2 line as a result of 24 hour salt stress. Similar results were obtained from two dimensional analysis ofin vivo labeled polypeptides. At least one isolated cDNA clone shows selective expression of mRNA in salt tolerant cells grown in NaCl. These results indicate that adaptive mechanisms for salt tolerance may differ in some aspects from acute stress mechanisms.     

Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy

A method has been developed to determine the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the protoplasts released from such cells by enzymatic digestion. It is demonstrated that there is a smooth and reproducible transition in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of protoplast release during enzymatic digestion of cell wall material.Abbreviations FTIR Fourier Transform Infrared - SEM Scanning Electron Microscope     

24-Epibrassinolide-induced alterations in the root cell walls of <Emphasis Type="Italic">Cucumis sativus</Emphasis> L. under Ca(NO<Subscript>3</Subscript>)<Subscript>2</Subscript> stress

Brassinosteroids (BRs) can effectively alleviate the oxidative stress caused by Ca(NO₃)₂ in cucumber seedlings. The root system is an essential organ in plants due to its roles in physical anchorage, water and nutrient uptake, and metabolite synthesis and storage. In this study, 24-epibrassinolide (EBL) was applied to the cucumber seedling roots under Ca(NO₃)₂ stress, and the resulting chemical and anatomical changes were characterized to investigate the roles of BRs in alleviating salinity stress. Ca(NO₃)₂ alone significantly induced changes in the components of cell wall, anatomical structure, and expression profiles of several lignin biosynthetic genes. Salt stress damaged several metabolic pathways, leading to cell wall reassemble. However, EBL promoted cell expansion and maintained optimum length of root system, alleviating the oxidative stress caused by Ca(NO₃)₂. The continuous transduction of EBL signal thickened the secondary cell wall of casparian band cells, thus resisting against ion toxicity and maintaining water transport.     

Dps proteins, an efficient detoxification and DNA protection machinery in the bacterial response to oxidative stress

The proteins belonging to the Dps (DNA-binding proteins from starved cells) family play an important role within the bacterial defence system against oxidative stress. They act on Fe(II) and hydrogen peroxide that are potentially toxic in the presence of air. Fe(II) forms spontaneously insoluble Fe(III) and reacts with molecular oxygen or its reduced forms to yield the highly damaging hydroxyl radicals. All Dps proteins have the distinctive capacity to annul the toxic combination of iron and hydrogen peroxide as they use the latter compound to oxidise Fe(II). In addition to this intrinsic DNA protection capacity, several members of the family, including the archetypical Escherichia coli Dps, protect DNA physically by shielding it in large Dps-DNA complexes. The structural and functional characteristics that endow Dps proteins with the chemical and physical protection mechanism are presented and discussed also in the framework of the varied situations that may be encountered in different bacterial species.      

Evidence for glycosylation on a DNA-binding protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

Background  

All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column.     

Ultrastructure of the cell wall of bromegrass (Bromus inermis Leyss) cell suspension culture

Thin sections of a bromegrass (Bromus inermis Leyss) cell suspension culture, examined electron microscopically, revealed unusual, often lens-shaped, spaces in cell walls. Freeze-fracture replicas of similar material demonstrated the presence of lamellae within the cell walls, both within comparable lens-shaped spaces and more extensively at cell wall surfaces. The cell wall also displayed strong yellow-green autofluorescence. It is proposed that lipidic material is present in these cell walls, the chemical identity of which is presently under investigation.     

Formation of anti-plant viral protein by Mirabilis jalapa L. cells in suspension culture

The extract of Mirabilis jalapa cultured cells and its precipitate fraction with 90% saturated ammonium sulfate showed an anti-plant viral activity comparable to that of the roots and leaves of the original plant. In the immunodiffusion experiment, the extract of cultured cells positively reacted with MAP (Mirabilis Anti-plant viral Protein) anti-serum. The changes in MAP formation during cell growth and the MAP content of roots and leaves were examined using enzyme-linked immunosorbent assay (ELISA). MAP formation proceeded almost in parallel with cell growth. The MAP content of cultured cells reached the highest level (0.6 mg/g dry weight) on the 9th day after inoculation, which was less than one-third of the content of the roots but three times larger than that of the leaves.Abbreviations MAP Mirabilis anti-plant viral protein - TMV tobacco mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - ELISA enzyme-linked immunosorbent assay Studies on the production of anti-plant viral substances of higher plant cells in suspension culture. Part 1