Specific intra-tissue responses to manganese in the floating lamina of Trapa natans L

Plant tolerance to heavy metals requires morpho-physiological mechanisms that are still poorly understood, especially in hydrophytes. This study focuses on the young floating lamina of the rhyzophyte Trapa natans exposed for 10 d to 130 microM Mn. The lamina has the ability to bioaccumulate Mn (> 3000 microg g(-1)). X-ray microanalysis of Mn cellular distribution revealed accumulation in the upper epidermis, in the first palisade layer, and in the idioblasts of the spongy tissue, which were shown with electron microscopy to contain osmiophilic vacuolar deposits, also observed to a minor extent in the control leaves. On the basis of biochemical and histochemical tests, these deposits were attributed to phenolic compounds that were probably able to chelate Mn. Net photosynthesis, photosynthetic pigments, room temperature microspectrofluorimetric analyses, and ultrastructural studies of plastids were performed to evaluate the status of the photosynthetic apparatus. A greater development of thylakoid membranes was observed in plastids of the second palisade and spongy tissue, which, however, did not accumulate Mn. Only the spongy tissue experienced inadequate assembly of PS II, but this did not significantly influence the photosynthetic yield of the whole lamina. It was concluded that T. natans can optimise productivity in the presence of Mn by means of specific intra-tissue responses within the framework of the floating lamina.     

Morpho-physiological and biochemical responses in the floating lamina of Trapa natans exposed to molybdenum

The response to molybdenum (Mo) was studied in the metal-tolerant hydrophyte Trapa natans L. Previously, it was shown that the plant accumulates Mn in the floating lamina by means of phenolic compounds and responded with acclimation responses of the chloroplast. Since the involvement of phenolics has been proposed also in Mo resistance, we tested the response of T. natans to increasing doses (5, 50, 150, 600 µM) of Mo using the photosynthetic apparatus as an indicator of cellular stress. Only 5 μM Mo did not cause evident modifications with respect to controls. Conversely, 50 to 600 μM Mo induced progressively marked alterations of the lamina morphology. The chloroplast ultrastructure showed disorganisation of the thylakoid system, and correspondingly, the photosynthetic pigment pattern was altered with a fall-down in photosynthesis. Microspectrofluorimetry indicated alterations of photosystem II, with differences among the three cell layers (first and second palisade and spongy tissues). While the highest dose caused plant death, 50 and 150 μM Mo-treated plants underwent partial recovery, and the plant survived up to the end of the vegetative season. However, reproduction was unsuccessful. Mo treatment did not induce increase in total phenolics, but only in anthocyanin. In contrast to Mn, detoxification of Mo by chelation inside vacuoles, possibly by anthocyanins, is suggested to be an insufficient mechanism to reduce Mo toxicity, which probably includes an impairment of nitrogen metabolism. However, the metal was accumulated in the lamina. On the whole, T. natans showed limited capabilities to survive Mo excess as compared with Mn.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Sugarcane micropropagation and phenolic excretion

Sugarcane shoot formation was followed using a temporary immersion system. Plant fresh weight, plant dry weight, shoot number and phenolic excretion to the culture medium were recorded during shoot formation. Shoot number increased for 30 days of culture but formation of new shoots was greatly reduced from 31 to 40 days. Phenolic excretion also increased during the first 20 days of culture (gallic acid represented 82% total phenolics) and decreased during the last 10 days (31–40 days of culture). The most intensive period of phenolic excretion (11–20 days) preceded the most intensive period of shoot formation (21–30 days). The same relationship does not seem to exist between the accumulation of fresh and dry weights. Subculture onto fresh medium at the beginning of proliferation (10 days after culture initiation) was detrimental to shoot formation in the subsequent period (11–20 days). However, such a detrimental effect could be avoided if gallic acid was added to the medium. Addition of cysteine to the culture medium reduced both excretion of phenolics and shoot formation but not fresh weight. The use of temporary immersion systems, the increase of culture medium volume per initial explant and the addition of paclobutrazol promoted both phenolic excretion and sugarcane shoot formation. Results presented here indicate a relationship between phenolic excretion and shoot formation but not with accumulation of plant weight. This revised version was published online in June 2006 with corrections to the Cover Date.     

Temporal and positional relationships between Mn uptake and low-pH-induced root hair formation in Lactuca sativa cv. Grand Rapids seedlings

We investigated whether low-pH-induced manganese (Mn) deficiency causes low-pH-induced root hair formation in lettuce seedlings. Both the number and length of root hairs increased in 0 μM Mn (Mn-free) at pH 6 and decreased in 3 mM Mn (excess Mn) at pH 4 compared with the values in 10 μM Mn (normal Mn). These results indicate an inhibitory effect of Mn on both root hair initiation and elongation. The time dependency of root hair induction caused by Mn deficiency corresponded to that caused by low pH. Within 1 h after the pH of the culture medium was reduced from pH 6 to pH 4, the Mn uptake by roots decreased to 43% of that at pH 6. These results suggest that low-pH-induced Mn deficiency promotes root hair formation. At low pH, the rate of Mn uptake was reduced in areas >2 mm from the root tip. Roots with low-pH-induced root hairs still showed low Mn uptake during 3 h of incubation at pH 6. Therefore, the additional root hairs induced by low pH did not compensate for the low-pH-induced decrease in Mn uptake     

Effects of water level fluctuation on the growth ofNelumbo nucifera Gaertn. in Lake Kasumigaura,Japan

Investigations were made of the growth ofNelumbo nucifera, an aquatic higher plant, in a natural stand in Lake Kasumigaura. A rise of 1.0 m in the water level after a typhoon in August 1986 caused a subsequent decrease in biomass ofN. nucifera from the maximum of 291 g d.w. m⁻² in July to a minimum of 75 g d.w. m⁻². The biomass recovered thereafter in shallower regions. The underground biomass in October tended to increase toward the shore. The total leaf area index (LAI) is the sum of LAI of floating leaves and emergent leaves. The maximum total LAI was 1.3 and 2.8 m² m⁻² in 1986 and 1987, respectively. LAI of floating leaves did not exceed 1 m² m⁻². The elongation rates of the petiole of floating and emergent leaves just after unrolling were 2.6 and 3.4 cm day⁻¹, respectively. The sudden rise in water level (25 cm day⁻¹) after the typhoon in August 1986 caused drowning and subsequent decomposition of the mature leaves. Only the young leaves were able to elongate, allowing their laminae to reach the water surface. The fluctuation in water level, characterized by the amplitude and duration of flooding and the time of flooding in the life cycle, is an important factor determining the growth and survival ofN. nucifera in Lake Kasumigaura.     

Free and glucosylated phenolics, phenol β-glucosyltransferase activity and membrane permeability in cucumber roots affected by derivatives of cinnamic and benzoic acids

Seven-day-old seedlings of cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.01, 0.1 and 0.5 mM solutions of derivatives of cinnamic acid (ferulic and p-coumaric acids) and benzoic acid (p-hydroxybenzoic and vanillic acids) as stress factors. In cucumber roots phenolics (free and glucosylated), phenol β-glucosyltransferase (E.C. 2.4.1.35) activity as well as membrane permeability were examined. The most intensive glucosylation took place in the first hour of stress duration in roots treated with 0.01 mM ferulic and p-coumaric acids and with 0.01 and 0.1 mM p-hydroxybenzoic and vanillic acids. At these concentrations a high phenol β-glucosyltransferase activity was found. The deterioration of capacity for phenolic glucosylation as well as the decrease of the phenol β-glucosyltransferase was observed at the higher concentrations. It was associated with increased membrane permeability.     

Regulation of Peroxidase-Dependent Oxidation of Phenolics in the Apoplast of Spinach Leaves by Ascorbate

The aqueous phase of the cell walls inside leaves (apoplast)of spinach contained ascorbate (AA) and dehydroascorbate (DHA).Ratios of AA to AA plus DHA were between 0.4 and 0.9, whereasthose inside leaves were higher than 0.9. The amounts of AAplus DHA in the apoplast were between 15 and 60 nmol (g fr wt)^–1of leaves. If the volume of the apoplast is about 10% of totalvolume of leaf cells, the concentrations of AA plus DHA werebetween 0.15 and 0.6 mM. Apoplastic AA was oxidized by hydrogenperoxide, and the oxidation was stimulated by phenolics suchas caffeic acid or ferulic acid by a factor of 10, suggestingthe presence in apoplast of peroxidases which are differentfrom AA peroxidase. The stimulation was due to the oxidationof AA by the primary oxidation products of phenolics with apoplasticperoxidase. Based on the data, the physiological significanceof the occurrence of AA in the apoplast is discussed in relationto the regulation of the apoplastic oxidation of phenolics. (Received January 8, 1992; Accepted February 28, 1992)     

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Summary. Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10mM MnSO₄. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion–tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.Correspondence and reprints: Dipartimento delle Risorse Naturali e Culturali, Università degli Studi di Ferrara, Corso Porta Mare 2, 44100 Ferrara, Italy.     

Enhancing antioxidative capacity of <Emphasis Type="Italic">Lepidium meyenii</Emphasis> calli by addition of methyl salicylate to culture medium

Addition of 0.01–0.2 mM methyl salicylate (MeSA) to the culture media, the antioxidative capacities of maca fresh calli were increased and were 1.19–1.88 times of the control. But MeSA inhibited maca cell growth and this negative effect was dose-dependent. The dry weights of maca calli were 0.60–0.95 times of the control when addition of 0.01–0.2 mM MeSA to the culture media. Elicited by 0.05 mM MeSA, the total phenolic contents in maca calli increased and were 1.25–1.37 times of those in the control in the period from the 10th to the 20th culture day. H₂O₂ and malondialdehyde (MDA) contents increased as well. At the same time, the activities of antioxidative enzymes had corresponding changes. In conclusions, the antioxidative capacity of maca calli could be enhanced by addition of MeSA to the culture media. Considering the increase of antioxidative capacity and inhibition of maca cell growth, 0.05 mM MeSA was optimal concentration.     

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 μg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 μg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 μM Fe²⁺ to this medium increased the yield to 226 μg/g DCW.     

Acetylene reduction in gnotobiotic cultures with rhizosphere bacteria and wheat

Summary In comparison with other crop species, sunflower (Helianthus annuus L.) has been found to be very tolerant of high manganese (Mn) concentrations in nutrient solution. Furthermore, sunflower was able to accumulate high Mn concentrations in plant tops without apparent detrimental effect on growth. The first symptom of excess Mn supply (c. 30M Mn in solution) was the appearance of small, dark-brown to black spots (<0.5 mm in diameter) on lower stems and on petioles and blades of the lower leaves. The spots were not necrotic and were visibly associated with the trichomes on these plant parts. Electron microprobe techniques demonstrated an accumulation of Mn in and around the trichomes. A compartmentation mechanism is suggested whereby sunflower is able to tolerate high Mn concentrations in its tissues through localization of Mn in a metabolically inactive form.At Mn concentrations approximately 6 times higher than that required to produce the small, dark spots, the upper recently-expanded leaves developed a veinal chlorosis and severe leaf crinkling of the interveinal areas. Dark brown lesions (>2 mm in size) developed on the lower leaves, especially along the veins. A concentration of 2205 g Mn g^–1 in the tops was associated with a 10% reduction in plant dry matter yield.     

Effect of medium-pH and MES on adventitious root formation from stem disks of apple

We have examined the effect of medium-pH on rooting using 1-mm slices cut from stems of apple microshoots. Before autoclaving, the pH of the rooting medium was set at various pH values between 4.5 and 8.0. During autoclaving, the pH drifted in particular in the alkaline region. Additional changes occurred during culture and the range set at 4.5–8.0 had shifted to 5.2–6.0 after autoclaving and 3 weeks of culture. When 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) had been added as buffering agent, the pH was stable when set at 5.0–6.5. Highest rooting was achieved at pH ~5.3 with and without MES (pH measured after autoclaving). This maximum did not correlate with highest auxin uptake. MES inhibited adventitious root formation during the initial phase of root formation when the meristemoids are being formed (ca. 30% reduction at 10 mM) but was promotive during outgrowth of the meristemoids to roots (30% increase at 10 mM). Inhibition and promotion by MES were not related to its buffering action as they were observed at all pHs.     

Physical environment in non-ventilated culture vessels affects <Emphasis Type="Italic">in vitro</Emphasis> growth and morphogenesis of several cultivars of <Emphasis Type="Italic">Dianthus Caryophyllus</Emphasis> L.

Summary Differences were shown in the environmental conditions inside four types of non-ventilated culture vessels (glass baby-food jars capped with metal steel or Magenta B-caps, jam glass jars capped with metal steel caps, and Magenta GA7 culture vessels) that were incubated in the same external growth room conditions. Vessel light transmittance varied from 83% to 53% and determined the availability of photosynthetic photon flux density (PPFD) for explants. The mean of medium desiccation in the culture period (30 d) was from 2.8 g in vessels with the lowest rate of gas exchange (0.02 h⁻¹) to 10.4 g in vessels with the highest (1.1 h⁻¹). In all cases, the temperature inside the culture vessel increased during the light period and decreased during the dark period, and relative humidity was close to 100%. Results of in vitro growth and morphogenesis of Dianthus caryophyllus L. evs. Scania, White Sim, Angeline, and Pink Calypso provided evidence that the environmental differences detected inside these four types of non-ventilated culture vessels was sufficient to affect micropropagation, mainly related with the specific sensitivity of each cultivar to the gas exchange and medium desiccation determined by the vessel type.     

Nonglandular leaf trichomes as short-term inducible defense of the grey alder,Alnus incana (L.), against the chrysomelid beetle,Agelastica alni L.

Summary The grey alder compensates leaf area losses due to insect grazing by continuously producing new leaves throughout the vegetative period. Different degrees of defoliation were attained experimentally by a controlled release of the oligophagous beetle Agelastica alni on arbitrarily selected trees from a homogenous population of young alders. The reduction in leaf area per tree significantly influenced the density of leaf trichomes, assessed 10–30 days later, on newly sprouting leaves only. Cross-correlations between leaf area reduction and trichome density were strongest for leaves which completed unfolding 14–21 days after damage. Dualchoice assays suggested a negative influence of trichomes on oviposition rate of A. alni. Removal of trichomes by shaving demonstrated the highly significant effect of trichomes on feeding behavior of adults and larvae in dual-choice assays. The role of the induced increase in trichome density as a possible short-term defense reaction against herbivorous insects is discussed.     

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease

19 F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with K _d values of  ≈ 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with K _d  ≈ 2 mM. Mn(II) paramagnetic broadening in ¹⁹F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease. Received: 30 March 1999 / Accepted: 28 September 1999     

Two-stage culture for producing berberine by cell suspension and shoot cultures of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

In vitro cultures of Berberis buxifolia were established using thidiazuron (4.5, 23 and 45 mM) or picloram (4 and 40 mM) as plant growth regulators for sustaining growth. For producing berberine, a two-stage culture was performed. In the first step, thidiazuron or picloram were used for biomass production followed by the production stage where benzylaminopurine (4.4 mM) was added as a plant growth regulator. Berberine yields (102 mg g⁻¹ DW) and in vitro shoot cultures (200 mg g⁻¹ DW) were significantly lower than those of whole plants in the field (416 mg g⁻¹ DW). The highest productivity (0.18 mg 1⁻¹ day⁻¹) was attained using picloram (either 4 on 40 mM) in the first stage for producing biomass.     

Early silicification of leaves and roots of seedlings of a panicoid grass grown under different conditions: anatomical relations and structural role

• Grasses accumulate high amounts of silica deposits in tissues of all their organs, especially at mature stage. However, when and under which conditions do grass seedlings begin to produce these silica deposits and their relation with anatomy and development is little known. Here we investigated the silicification process in the first leaves and roots of seedlings of Bothriochloa laguroides grown in different substrate and Si treatments.
• The distribution and content of silica deposits in the organs of the seedlings grown under different conditions were analyzed through staining techniques and SEM‐EDAX analyses.
• Leaf silica deposits were accumulated 3–4 days after the first leaf emergence, also under low silica solution (0.17–0.2 mM). Their location was mainly restricted to short costal cells from basal sectors, and scarcely in trichomes and xylem at tips. Silica content in leaves increased with the age of the seedlings. Roots presented dome‐shaped silica aggregates, between 4–12 μm of diameter, located in the inner tangential wall of endodermal cells and similar to those produced at maturity.
• Silicification begins early in the first photosynthetic leaf, and silica distribution is opposite to that found in mature plants, mainly restricted to basal sectors, probably acting as a reinforcing element. The fast incorporation of solid amorphous silica in leaves and roots, may be useful for farm applications in species that are Si‐fertilized.

     

Effects of salinity on photosynthetic characteristics in photomixotrophic cell-suspension cultures from Alternanthera philoxeroides

We studied the effect of NaCl salinity on the development of cellular photosynthesis using a green, photomixotrophic, cell-suspension culture of Alternanthera philoxeroides (Mart.) Griseb. For these cells, increasing the concentration of sucrose in the media produces a rapid drop in net photosynthetic rate, which recovers as sucrose is depleted from the media. This predictable recovery provides a simple system to examine cellular photosynthetic development. Cells, unadapted to high salinity, were transferred to nutrient media with 30 mM sucrose (Control) or nutrient media with 30 mM sucrose and 100 mM NaCl (Salt). A dramatic increase in the dark respiration rate of Control and Salt cells during the first 6 d of the experiment produced net oxygen consumption in the light. The high dark respiration rates during this period were accompanied by a decline in total Chl and the amounts of two photosynthetic proteins, the light harvesting Chl a/b binding protein of photosystem II (LHCP) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco SSU). The dark respiration rate of Salt cells was greater than that of Control cells on days 4–8. After day 4, dark respiration rates decreased and net photosynthesis increased to stable values in both treatments at day 11 after media sucrose concentration reached a minimum. As dark respiration rates decreased and net photosynthetic rates increased, total Chl and the amounts of LHCP and rubisco SSU increased in both Control and Salt cells. The slower development of photosynthetic capacity in salt cells was correlated with a fresh weight that was 20% lower than that of control cells at the end of the experiment.     

Coal Dust Pollution Effects on Wetland Tree Species in Richards Bay,South Africa

In this study, the effects of coal dust on four, sympatric, wetland tree species in Richards Bay Harbour were investigated. We tested the hypothesis that leaf micromorphology influenced dust accumulation and that coal dust occluded stomata and reduced photosynthetic performance of three mangroves, Avicennia marina, Bruguiera gymnorrhiza and Rhizophora mucronata, and a mangrove associate, Hibiscus tiliaceus. To investigate leaf micromorphology, leaf blade material of the four species was prepared following standard procedures and viewed under scanning electron microscopy. Gas exchange and chlorophyll fluorescence measurements were made at saturating light (>1000 μmol m⁻² s⁻¹) and high temperature (>25 °C) on leaves that were either covered or uncovered with coal dust. There was no evidence of occlusion of stomata by dust. Dust accumulation in A. marina and H. tiliaceus was exacerbated by the presence of a dense mat of trichomes on the undersurface of the leaves, as well as by the sticky brine secreted by salt glands in the former species.Coal dust significantly reduced CO₂ exchange, Photosystem II (PS II) quantum yield and electron transport rate (ETR) through PS II in A. marina and H. tiliaceus but not in the other two mangroves. Reduction in photosynthetic performance was attributed to reduction in light energy incident on the photosynthetic tissues.