Antagonism of Lactic Acid Bacteria against Phytopathogenic Bacteria

A variety of lactic acid bacteria, isolated from plant surfaces and plant-associated products, were found to be antagonistic to test strains of the phytopathogens Xanthomonas campestris, Erwinia carotovora, and Pseudomonas syringae. Effective “in vitro” inhibition was found both on agar plates and in broth cultures. In pot trials, treatment of bean plants with a Lactobacillus plantarum strain before inoculation with P. syringae caused a significant reduction of the disease incidence.     

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon

The extent and occurrence of water stress-induced “patchy” CO₂ uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [¹⁴C]CO₂ before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to −1.1 megapascals during a 4-day period did result in heterogenous CO₂ assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO₂ assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO₂] in stressed bean leaves. When extent of heterogenous CO₂ uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to −1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO₂ assimilation patterns when stressed to a leaf water potential of −2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.     

Uptake and distribution of N-phosphonomethylglycine in sugar beet plants

Glyphosate (N-phosphonomethylglycine) was readily transported in sugar beet plants (Beta vulgaris L., Klein E type, monogerm). Concentrations in sink leaves reached 2.5 to 13.7 micromolar in 10 hours from a 15 millimolar solution supplied to one mature leaf. Distribution of glyphosate followed that of [³H]sucrose used as a marker for materials transported by phloem, indicating that this is the primary means for distribution of glyphosate. Possible mechanisms of entry into the sieve tubes were evaluated using isolated leaf discs. Concentration dependence of uptake and kinetics of exodiffusion from tissue indicate a passive, nonfacilitated mechanism. Uptake was not affected by pH, eliminating the passive, weak acid mechanism. Permeability of the plasmalemma to glyphosate was calculated as 1.7 × 10⁻¹⁰ meters per second. This characteristic would allow slow entry and exit from the phloem, and together with other physiological parameters of the plant, is postulated to allow accumulation and transport in the phloem.     

Quantitative analysis of transpiration stream dynamics in an intact cucumber stem by a heat flux control method

Water flux of transpiration stream in an intact stem of the 10 leaf stage cucumber plant (Cucumis sativus L. cv. Chojitsu-Ochiai) was measured by a novel system of heat flux control method with a resolution of 1 × 10⁻³ grams per second and a time constant of 1 minute; two heat flux control sensors were attached to the seventh internode and the stem base. The transpiration stream responded clearly to leaf transpiration and root water absorption when the plant was exposed to light, and the water flux at the stem base corresponded to the transpiration rate per plant in steady state. Root water absorption lagged about 10 minutes behind leaf transpiration. Dynamics of water fluxes were affected by the lag of water absorption in roots, and temporary water loss caused by rapid increase in leaf transpiration was buffered by about 5% of the water content in the stem.     

Xanthomonas axonopodis pv. phaseoli var. fuscans Is Aggregated in Stable Biofilm Population Sizes in the Phyllosphere of Field-Grown Beans

The occurrence of “Xanthomonas axonopodis pv. phaseoli var. fuscans” (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10⁵ CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.     

Effect of Light and Chilling Temperatures on Chilling-sensitive and Chilling-resistant Plants. Pretreatment of Cucumber and Spinach Thylakoids in Vivo and in Vitro

The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. “Marketer”) and spinach (Spinacia oleracea L. “Bloomsdale”) were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity and chlorophyll is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light.     

Involvement of phaseolotoxin in halo blight of beans: transport and conversion to functional toxin

Phaseolotoxin ([N^δ-phosphosulfamyl]ornithylalanylhomoarginine) is produced by Pseudomonas phaseolicola (Burkh.) Dows. in liquid culture. When phaseolotoxin was applied to leaves of bean (Phaseolus vulgaris L.) at 0.1 to 1 nmoles/g fresh weight of leaf by a prick-assay procedure, the characteristic “halo” symptom of bean halo blight disease developed after 24 to 48 hours. At higher concentrations (10-100 nmoles/g fresh weight) the systemic symptoms, which are commonly a feature of diseased plants, also developed after 24 to 48 hours.     

Stimulation of growth and ion uptake in bean leaves by red and blue light

Red and blue light both stimulate growth and ion accumulation in bean (Phaseolus vulgaris L.) leaves, and previous studies showed that the growth response is mediated by phytochrome and a blue-light receptor. Results of this study confirm that there is an additional photosynthetic contribution from the growing cells that supports ion uptake and growth. Disc expansion in the light was enhanced by exogenous K⁺ and Rb⁺, but was not specific for anions. Light increased K⁺ accumulation and the rate of ⁸⁶Rb⁺ uptake by discs, over darkness, with no effect of light quality. The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, inhibited light-driven ⁸⁶Rb⁺ uptake by 75%. Light quality caused differences in short-term kinetics of growth and acidification of the leaf surface. At comparable fluence rates (50 μmol m⁻² s⁻¹), continuous exposure to blue light increased the growth rate 3-fold after a 2-min lag, whereas red light caused a smaller growth response after a lag of 12 min. In contrast, the acidification of the leaf surface normally associated with growth was stimulated 3-fold by red light but only slightly (1.3-fold) by blue light. This result shows that, in addition to acidification caused by red light, a second mechanism specifically stimulated by blue light is normally functioning in light-driven leaf growth.     

Isolation of Mesophyll Cells from Sedum telephium Leaves

A technique is described for mechanically isolating metabolically active individual spongy mesophyll cells from the Crassulacean acid metabolism plant, Sedum telephium. Mature leaves are selected at about 2 PM when acidity is low, and three different media are used to reduce the problem of leaf acidity and to maintain isotonic conditions. The upper and lower epidermis is peeled from chilled leaves and the leaves are suspended in a buffered “soaking medium,” then gently ground with a mortar and pestle. Cells and debris are separated using a “washing medium,” with cells being filtered through a 136 micron net and collected on an 80 micron net. Cells then are suspended in a “cell suspension medium” and concentrated by centrifugation. Approximately 2 hours are required for the isolation procedure, and activity in CO₂ fixation is constant for up to 4 hours after isolation. Microscopic examination shows about 65% of the isolated cells appear intact and unplasmolyzed and are similar to leaf msophyll cells. The yield of cells on a leaf chlorophyll basis is about 1%. A light micrograph of the isolated cells is given.     

Effect of Fruits on Dormancy and Abscisic Acid Concentration in the Axillary Buds of Phaseolus vulgaris L

The mechanism regulating the growth of adult plants in two determinate bean (Phaseolus vulgaris L.) cultivars was investigated. “Redkloud” plants flowered, formed fruits, and ceased shoot growth earlier than “Redkote” plants. Redkloud attained a smaller plant size, compared to Redkote, by imposing dormancy on axillary buds at an earlier age. In both cultivars, cessation of bud growth coincided with maximum combined fruit length per plant. Removal of fruits caused resumption of axillary bud growth within 4 to 5 days. The amount of new growth induced by fruit removal depended on the cultivar and plant age. In fully developed Redkloud plants, where shoot growth had already ceased, total leaf and shoot number per plant nearly doubled within 2 weeks following fruit removal. A much smaller response was observed in the still growing Redkote plants. Fruits, therefore, are assumed to play a major role in the regulation of shoot growth and total plant size through the control of axillary bud dormancy. It seems that smaller plant size, earlier maturity, and earlier senescence of Redkloud, compared to Redkote, were the result of earlier flowering, and accomplished in part through the growth-inhibiting action of fruits.     

Effects of glyphosate on the movement of assimilates of two Lolium perenne L. populations with differential herbicide sensitivity

Glyphosate applications trigger the depletion of aromatic amino acid pools and the decrease of photosynthesis that results in changes in carbon metabolism. The aim of this work was to determine the effect of glyphosate on the export of ¹⁴C from ¹⁴C-glucose to the main sinks, by comparing a glyphosate-resistant Lolium perenne population with a susceptible one. Untreated plants of the two populations grown in hydroponics were labeled with ¹⁴C-glucose applied at the youngest expanded leaf at the tillering stage. Similar ¹⁴C-glucose absorption and ¹⁴C distribution patterns were recorded in both populations. In another experiment, half of the plants of each population were treated with glyphosate, whereas the other half was sprayed with water (controls). Glucose absorption did not vary under glyphosate treatment, regardless of the sensitivity of each population to the herbicide. However, the translocation of ¹⁴C and its distribution patterns were significantly affected by glyphosate within 1 day in the susceptible population. The treated susceptible plants showed 57% higher ¹⁴C retention at the labeled area than their controls. The lower ¹⁴C movement significantly affected the unexpanded leaves and the apical meristem on the labeled tiller. Moreover, the ¹⁴C released from roots was significantly decreased by glyphosate only in the susceptible plants. Glyphosate did not influence leaf absorption, translocation, or release of ¹⁴C-labeled glucose plus radiolabeled metabolites in the resistant population.     

The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene

Maize (Zea mays L. var. Bonnie) transformed with a gene encoding a 5-enolpyruvylshikimate 3-phosphate synthase with altered sensitivity showed over 100-fold greater resistance to the herbicide glyphosate (N-[phosphonomethyl]glycine) in comparison with its non-transformed progenitor (parental control) at the third-leaf stage. Studies with [¹⁴C]-glyphosate at a dosage lethal to the parental control, but sublethal to the transgenic, revealed that a maximum of 45-65% of the applied dose was absorbed, with greater absorption occurring in transgenic plants. Translocation of glyphosate was closely related to its absorption (r value 0.956) with approximately 15% more of the applied dose being mobilized in transgenic plants than the parental controls. Analysis of electronic autoradiograms along the treated leaf lamina found discrete internal regions of glyphosate accumulation closely associated with the site of application. These regions contained lower amounts of glyphosate present in the treated leaf lamina was almost completely translocated in transgenic plants, while in the parental controls more remained and the leaf became necrotic. In both types of maize there was a small accumulation of herbicide in the tip region of the leaf which was not mobilized. Younger shoot tissues and roots were major sinks for translocated glyphosate accumulating approximately 25-40% of the applied dose depending upon treatment. In the parental control, equal amounts of glyphosate were found distributed between young shoot tissues and roots; while in transgenic plants, the young shoot tissue accumulated around three times more glyphosate than the roots. In both plant types, glyphosate was localized in the meristems and young, actively growing leaves. Specific glyphosate activity (the amount of glyphosate per unit dry weight of tissue) in the major sinks of the transgenic declined towards the end of the treatment period but remained relatively constant in the parental control. In conclusion, enhancing glyphosate resistance by genetic transformation influenced the absorption, translocation and distribution of this herbicide in whole plants.Keywords: Zea mays, glyphosate (N-[phosphonomethyl]-glycine), transgenic, absorption, translocation, source-sink.      

TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus

In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K⁺ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K⁺ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K⁺ efflux. NcTOKA channel gating was sensitive to extracellular K⁺ such that channel activation was dependent on the reversal potential for K⁺. However, expression of NcTOKA was able to overcome the K⁺ auxotrophy of a yeast mutant missing the K⁺ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K⁺ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K⁺ currents at potentials negative of E_K. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca²⁺, verapamil, quinine, and TEA⁺ but was insensitive to Cs⁺, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.     

Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth

Root temperature strongly affects shoot growth, possibly via “nonhydraulic messengers” from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25° and 35°C, respectively. Hydraulic conductance (L_p) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25°C, remaining high with further warming. In sorghum, the L_p of intact plants and of detached roots peaked at 35°C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L_p. Extreme temperatures greatly decreased ion flux, particularly K⁺ and NO₃⁻, to the xylem of detached roots of both species. Removing external K⁺ did not alter short-term K⁺ flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently “uncoupled” from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.     

A mechanism for the leaching of calcium from foliage

Young bean plants (Phaseolus vulgaris) containing root-absorbed ⁴⁵Ca and ⁸⁶Rb were leached to determine the pathway and mechanism of cation loss by leaching. Calcium is leached from the exchangeable calcium fraction within the plant by a process of ion exchange and diffusion involving exchange sites both within the leaf and on the leaf surface. Leaching of cations is primarily a passive process, although some metabolites may be deposited upon leaf surfaces by active processes. The exchange and diffusion explanation is compatible with current theories of ion uptake and translocation and explains the results of numerous experiments on leaching reported in the literature.     

Subcellular Localization of Zinc and Calcium in Bean (Phaseolus vulgaris L.) Tissues

Two bean (Phaseolus vulgaris L.) cultivars differing in growth responses to zinc were examined for differences in uptake and subcellular localization of ⁶⁵Zn during a 15-day growth period. The zinc-sensitive cultivar Sanilac showed initially a much higher rate of absorption, which declined after 24 hours. The zinc-tolerant cultivar Saginaw showed a slow but steady rate of absorption for 10 days. In roots as well as in stem callus tissues of both cultivars, three-fourths of the absorbed ⁶⁵Zn was localized in the “cytoplasmic” supernatant fractions (containing ribosomes and vacuolar sap). Very little (less than 7%) ⁶⁵Zn was localized in the cell wall fraction. There was a much greater proportion of the absorbed ⁶⁵Zn localized in root mitochondria and nuclei of the zinc-sensitive Sanilac than in the zinc-tolerant Saginaw. Stem callus tissues, however, did not show such cultivar differences in zinc accumulation at the sub-cellular level.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

Euploidy in Ricinus: EUPLOIDY EFFECTS ON PHOTOSYNTHETIC ACTIVITY AND CONTENT OF CHLOROPHYLL-PROTEINS

The effects of nuclear genome duplication on the chlorophyll-protein content and photochemical activity of chloroplasts, and photosynthetic rates in leaf tissue, have been evaluated in haploid, diploid, and tetraploid individuals of the castor bean, Ricinus communis L. Analysis of this euploid series revealed that both photosystem II (2,6-dichlorophenolindophenol reduction) and photosystem I oxygen uptake (N,N,N′,N′-tetramethyl-p-phenylenediamine to methyl viologen) decrease in plastids isolated from cells with increasingly larger nuclear complement sizes. Photosynthetic O₂-evolution and ¹⁴CO₂-fixation rates in leaf tissue from haploid, diploid, and tetraploid individuals were also found to decrease with the increase in size of the nuclear genome. Six chlorophyll-protein complexes, in addition to a zone of detergent complexed free pigment, were resolved from sodium dodecyl sulfate-solubilized thylakoid membranes from cells of all three ploidy levels. In addition to the P700-chlorophyll a-protein complex and the light-harvesting chlorophyll a/b-protein complex, four minor complexes were revealed, two containing only chlorophyll a and two containing both chlorophyll a and b. The relative distribution of chlorophyll among the resolved chlorophyll-protein complexes and free pigment was found to be similar for all three ploidy levels.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the K_m was 7.1 μM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.