Immunogold localisation of hemoglobin inCasuarina root nodules

Summary Immunocytochemical localisation of hemoglobin on sections ofFrankia-infecledCasuarina glauca nodule tissue confirmed its presence in nitrogen-fixing infected cells. Using colloidal gold as the marker, hemoglobin was shown to be restricted to the cytoplasm and nucleus of infected cells. None was associated with endophyte hyphae or uninfected cells. As infection developed, with its associated thickening and modification of host cell walls, the level of label, and by implication, the level of hemoglobin increased.Abbreviations SPL semi-permeable layer - GA glutaraldehyde - PFA paraformaldehyde     

Photosynthetic carbon sources in somepotamogeton species

The ability of somePotamogeton species to use bicarbonate for photosynthesis was assayed by means of relative net photosynthetic rates in alkaline (bicarbonate) and acid (free CO₂) solutions with an equivalent amount of inorganic carbon. The results showedPotamogeton crispus, P. oxyphyllus, P. maackianus, P. perfoliatus, P. malaianus and the submerged leaves ofP. distinctus to be able to use bicarbonate ions for photosynthesis. To the contrary, the submerged leaves ofP. fryeri, which is an inhabitant of soft and acid waters, and the floating leaves ofP. distinctus were proven to be ‘non-users’ of bicarbonate ions. Furthermore, the relative photosynthetic rates were determined using natural waters with various carbon conditions. The dependence of photosynthetic performance on the free CO₂, pH and alkalinity of the waters is discussed in relation to carbon metabolism. Further it is suggested that the carbon conditions are ecologically significant in relation to the productivity and success of macrophyte species in natural habitats.     

Beeinflussung der Blattzellen vonFontinalis antipyretica durch den Bewuchs vonCocconeis placentula

In a special habitat near Lunz (Mittersee) long time epiphytism ofCocconeis placentula var.tenuistriata and var.klinoraphis onFontinalis antipyretica leaves causes characteristic, strongly localized alterations in the walls of affected cells. They turn brown, may thicken, and may produce cystolithe-like protuberances into the cell lumina. These reactions obviously are brought about by a specific property ofCocconeis, for other epiphytes do not cause any change. The comparison of theCocconeis vegetation on younger and olderFontinalis leaves shows a considerable variability of the reactions mentioned, and in another habitat none could be found. Apparently several factors cause these reactions. Altogether,Cocconeis placentula is not a completely harmless epiphyte under certain circumstances. The alterations of the moss cells may be interpreted partly as a premature aging process, partly as a defensive action corresponding to alterations induced by some parasitic fungi. Furthermore, the reactions ofCocconeis onFontinalis are remarkably parallel to the influence of the dinococcalean algaRaciborskia inermis onSpirogyra grevilleana.

     

Lipid polymers accumulate in the epidermis and mestome sheath cell walls during low temperature development of winter rye leaves

Summary Winter rye (Secale cereale L cv. Puma) was grown at 20 °C and at 5 °C and the development of epidermal and mestome sheath cells of leaves from plants grown at both temperatures was compared by electron microscopy. At 5 °C, the cells became densely packed with cytoplasm and small vacuoles after 41 days of growth. By day 56 at 5 °C, epidermal and mestome sheath cells were small in diameter and multivacuolate with asymmetrically thickened walls. By day 76 at 5 °C, a new developmental stage had been reached in epidermal and mestome sheath cells. The cells were larger in diameter although the thickened cell walls and multivacuolate cytoplasm were still present. As epidermal and mestome sheath cell walls thickened during low temperature growth of winter rye, an increase in cuticle thickness and the deposition of a lamellar layer could be observed in epidermal and mestome sheath cells, respectively. The lipid-derived polymers from the leaves of rye plants grown at 20 °C were shown by reductive depolymerization and GC-MS analysis to be comprised of 18-hydroxy-9, 10-epoxyoctadecanoic acid (47%) and dihydroxyhexa-decanoic acid (29%). The leaves of plants grown at 5 °C had two to four times as much lipid-derived polymeric material as those grown at 20 °C and the proportion of the major monomer, 18-hydroxy-9,10-epoxyoctadecanoic acid, increased to 73% of the polymeric material. Physical isolation of both epidermal tissue and vascular bundles followed by GC-MS analysis of the monomeric components released by reduction of the respective lipid polymers showed that 18-hydroxy-9,10 epoxyoctadecanoic acid was the major monomer in the polymer of both the epidermis and the mestome sheaths. The presence of this epoxide monomer in both the cuticles and mestome sheath cell walls of rye leaves was confirmed and visualized by using an epoxide-specific staining reaction.     

Fermentation of cheese whey by a mixed culture ofClostridium beijerinckii andBacillus cereus

Summary Fermentation of cheese whey to produce butanol and butyric acid was carried out using a mixed culture ofClostridium beijerinkii andBacillus cereus. Fermentation selectivities were studied by controlling the pH of the system. Controlled pH values higher than 6.5 as well as those below 5.0 were not conducive to butanol production. Maximum product formation was obtained by controlling the pH at 5.5. When compared with the results obtained using the pure culture ofC. beijerinckii, a higher butanol concentration was obtained in the mixed culture without sacrificing the level of butyric acid formed.     

Differential distribution of ascorbic acid and RNA in the developing anthers ofDatura stramonium L.

The histochemical localization of ascorbic acid and RNA was studied during developmental stages ofDatura anthers. The concentration of ascorbic acid and RNA was high in primary parietal and primary sporogenous layers, sporogenous cells and pollen grains. The connective of young anther showed remarkably high concentration of ascorbic acid. The high peaks of ascorbic acid and RNA concentration correlated with the growth phases of anther. The connective and anther wall layers act as reservoirs of energy needed for developing sporogenous cells.     

Some observations on cell wall structure and taxonomy ofPhymatodocis nordstedtiana (Conjugatophyceae,Chlorophyta)

Filaments ofPhymatodocis nordstedtiana Wolle were isolated from a sample of a Texan lake. Cultures were established and examined by light and scanning electron (SEM) as well as transmission electron microscopy (TEM). It is shown that the pores apparent on light microscopical examination are not of the cosmaroid type as expected. TEM examination disclosed that they are similar to those found in the generaClosterium Ralfs andPenium Bréb. Furthermore, it could be demonstrated by light and SEM microscopy that the primary cell wall is shed during cell division. The remaining secondary cell wall of the mature cell consists of interwoven bands of parallel microfibrils. A conspicuous overlap of the semicell walls clearly denotes the isthmus region. The significance of these deviations unusual for desmids is discussed. Suggestions are made that the taxonomic position ofPh. nordstedtiana should be re-evaluated.     

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

A stable reddish purple anthocyanin in the leaf ofGynura aurantiaca cv. ‘Purple Passion’

The anthocyanin (GAA) in the epidermis and hair of the leaf ofGynura aurantiaca cv. ‘Purple Passion’ was isolated and identified as cyanidin tetra-glucoside acylated by three molecules of caffeic acid and one molecule of malonic acid. GAA was also isolated from the lower epidermis of the leaf ofG. bicolor DC. GAA showed a very stable reddish purple color from weakly acid to neutral pH region, but the color of the deacylated compound disappeared rapidly in the same region. This indicated that the attached organic acids must play an essential role in the stabilization of the color. Comparison of the profiles of the visible absorption spectra of the intact epidermal peels and cells ofG. aurantiaca andG. bicolor with those of GAA dissolved in various pH solutions suggested that the pH of the epidermal vacuole containing GAA was nearly 4.3. GAA was indistinguishable from the anthocyanin (rubrocinerarin) which we had previously isolated from the purplish red flowers ofSenecio cruentus DC. by means of UV-Vis, NMR and Mass spectra. Deceased     

Open dichotomous venation in the leaves ofUtricularia striatula (Lentibulariaceae)

The open dichotomous pattern of venation in the leaves ofUtricularia striatula is described in detail and compared withCircaeaster andKingdonia. Similar traits of dichotomous venation occur in other species ofUtricularia and may be due to reduction.Studies on IndianUtricularia, 3.     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

The effects of Penicillium digitatum and Fusarium oxysporum rots on nutritional content of pawpaw (Carica papaya L.)

Investigations were carried out on the effects of Penicillium digitatum and Fusarium oxysporum on the nutritional value of pawpaw (Carica papaya). Decreases were observed in ash content, phosphorus, sodium, reducing sugars and ascorbic acid levels of fruits infected with P. digitatum, but increases in calcium and potassium content. In fruits infected by F. oxysporium, there were decreases in phosphorus, calcium, sodium ascorbic acid and reducing sugar levels; but the levels of ash content increased. The total protein level increased in the fruits infected with both fungi. These results revealed a reduction in fruit quality.     

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.     

Arthopyrenia endobrya,spec. nov., eine hepaticole Flechte mit intrazellulärem Thallus aus Brasilien

Arthopyrenia endobrya from Southern Brazil is illustrated and described as a new species of lichens. The thallus is composed of filamentous green algae loosely surrounded by fungal hyphae. Both symbionts grow endophytically within the leaf cells of two species ofLejeuneaceae (Hepaticae). The algae and hyphae penetrate the cell walls of the host by means of fine perforations. The ascocarps develop between the leaves and perforate them with their apical region. The classification as a member of the genusArthopyrenia is preliminary.

     

Pectate distribution and esterification in Dubautia leaves and soybean nodules,studied with a fluorescent hybridization probe

Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid) - NMR nuclear magnetic resonance spectroscopy - TTB 1,3,5-triazido-2,4,6-trinitrobenene     

Ultrastructural aspects of kranz anatomy inDigitaria sanguinalis andSetaria viridis (poaceae)

Structural differentiation of Kranz anatomy has been investigated in leaf cross sections of two C-4 Poaceae:Digitaria sanguinalis andSetaria viridis. The study mainly focused on cellular and interfacial features of bundle sheath (BS) and mesophyll (MS) cells of the C-4 structure. Prominent BS, spaced by only two MS cells apart, were surrounded concentrically by a layer of MS cells. BS cells ofS. viridis had centrifugally arranged relatively large chloroplasts containing much starch, but the chloroplasts had agrana to rudimentary grana. Structural and size dimorphisms, when starch was present, were detected between BS and MS chloroplasts. Loosely arranged MS cells had peripherally displaced smaller chloroplasts containing little to none starch. BS chloroplasts ofD. sanguinalis were similar to those ofS. viridis, but had very little starch and well-developed long agranal stroma lamella. Features of MS cells were similar in both species, but well-defined peripheral reticulum (PR) was easily recognized in MS chloroplasts ofS. viridis. Virtually no PR was developed in BS chloroplasts examined. BS cells contained more mitochondria and microbodies, but no structural dimorphism was noticed. The electron-dense suberized lamella were often observed between BS and MS cells, especially in the primary wall of BS cells. It was most frequently found at the BS and MS cell interfaces and terminated in radial walls of the adjacent BS cells. Prominent pits with plasmodesmata (pd) were seen in the walls of both cells. There also were numerous pd in outer tangential walls of the BS cells. The number of pd ranged from 20 to 60. The pd trasversed a segment of cell wall much thinner than the adjacent wall. The current cellular data have been compared to the ultrastructural features known in leaves of other C-4 plants, especially NADP-ME species.     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O₂ diffusion in nodules ofCasuarina cunninghamiana.     

Proton excretion and cell expansion in bean leaves

Light-induced expansion of Phaseolus vulgaris L. leaf cells is accompanied by increased cell-wall plasticity. The possibility that leaf-cell walls are loosened by excreted protons has been investigated. First, light causes acidification, detected at the leaf surface, within 5–15 min. Growth starts 10–20 min after exposure to light. Second, exogenous acid induces loosening of isolated leaf cell walls. Third, infiltration of the tissue with a neutral buffer inhibits light-induced growth. Fourth, fusicoccin stimulates growth of as well as H⁺ excretion by bean leaf cells, without light. These findings show that the acid-growth theory is applicable to light-induced growth of leaf cells, and indicate that light-induced proton excretion initiates cell enlargement in leaves.Abbreviations FC fusicoccin - RL red light - WEx wall extensibility - WL white light     

Impaired growth in transgenic plants over-expressing an expansin isoform

Expansins are cell wall proteins characterised by their ability to stimulate wall loosening during cell expansion. The expression of some expansin isoforms is clearly correlated with growth and the external application of expansins can stimulate cell expansion in vivo in several systems. We report here the expression of a heterologous expansin coding sequence in transgenic tomato plants (Lycopersicon esculentum Mill.) under the control of a constitutive promoter. In some transgenic lines with high levels of expansin activity extractable from cell walls, we observed alterations of growth: mature plants were stunted, with shorter leaves and internodes, and dark-grown seedlings had shorter and wider hypocotyls than their wild-type counterparts. Examination of hypocotyl sections revealed similar differences at the cellular level: cortical and epidermal cells were shorter and wider than those from wild-type seedlings. The observed stimulation of radial expansion did not compensate for the decreased elongation, and overall growth was reduced in the transgenics. As this observation can seem paradoxical given the known effect of expansins on isolated cell walls, we examined the mechanical behaviour of transgenic tissue. We measured a decrease in hypocotyl elongation in response to acidic pH in the transformants. This result may account for the alterations in cell expansion, and could itself be explained by a reduced susceptibility of transgenic cell walls to expansin action.