Interactive Effects of Abiotic Stress and Elevated CO2 on Physio-Chemical and Photosynthetic Responses in Suaeda Species

Suaeda fruticosa and S. monoica are important halophytes for ecological rehabilitation of saline lands. We report differential physio-chemical, photosynthetic, and chlorophyll fluorescence responses in these halophytes under 100 mM sodium chloride (NaCl), 50% strength (16.25 ppt) of seawater (SW)-imposed salinity, and 10% polyethylene glycol 6000 imposed osmotic stress at 380 (ambient) and 1200 (elevated) µmol mol^–1 CO₂ concentrations. SW salinity enhanced the growth in both species; however, compared with S. fruticosa, the S. monoica exhibited comparatively better growth and biomass accumulation under saline conditions at elevated CO₂. Results demonstrated better photosynthetic performances of S. monoica under stress conditions at both levels of CO₂, and this resulted in higher accumulation of carbon, nitrogen, sugar, and starch contents. S. monoica exhibited improved antenna size, electron transfer at PSII donor side, and efficient working of photosynthetic machinery at elevated CO₂, which might be due to efficient upstream utilization of reducing power to fix the CO₂. The δ¹³C results supported the operation of C₄ CO₂ fixation in S. monoica and C₃ or intermediate pathway of CO₂ fixation in S. fruticosa. Lower accumulation of reactive oxygen species, reduced membrane damage, lowered solute potential, and higher accumulation of proline and polyphenol contents indicated elevated CO₂-induced abiotic stress tolerance in Suaeda. Higher activity of antioxidant enzymes in both species at both levels of CO₂ help plants to combat the oxidative stress. Upregulation of NADP-dependent malic enzyme and NADP-dependent malate dehydrogenase genes indicated their role in abiotic stress tolerance as well as photosynthetic carbon (C) sequestration. Operation of C₄ type CO₂ fixation in S. monoica and an intermediate CO₂ fixation in S. fruticosa could be the possible reason for the superior photosynthetic efficiency of S. monoica under stress conditions at elevated CO₂.

     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

Residue Specific Ribose and Nucleobase Dynamics of the cUUCGg RNA Tetraloop Motif by MNMR 13C Relaxation

The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with ¹³C and ¹⁵N were studied by ¹³C spin relaxation experiments. R₁, R_1ρ and the ¹³C-{¹H} steady-state NOE of C₆ and C_1′ in pyrimidine and C₈ and C_1′ in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time τ_c of 2.31±0.13 ns and an axial ratio of 1.35±0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported ¹³C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539–10551; Fiala et al., 2000 J. Biomol. NMR 16, 291–302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165–190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S² < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S² = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of ¹³C relaxation rates were found to be in agreement with ¹⁵N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702–709). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots

The composition of suberin and lignin in endodermal cell walls (ECWs) and in rhizodermal/hypodermal cell walls (RHCWs) of developing primary maize (Zea mays L.) roots was analysed after depolymerisation of enzymatically isolated cell wall material. Absolute suberin amounts related to root length significantly increased from primary ECWs (Casparian strips) to secondary ECWs (suberin lamella). During further maturation of the endodermis, reaching the final tertiary developmental state characterised by the deposition of lignified secondary cell walls (u-shaped cell wall deposits), suberin amounts remained constant. Absolute amounts of lignin related to root length constantly increased throughout the change from primary to tertiary ECWs. The suberin of Casparian strips contained high amounts of carboxylic and 2-hydroxy acids, and differed substantially from the suberin of secondary and tertiary ECWs, which was dominated by high contents of ω-hydroxycarboxylic and 1,ω-dicarboxylic acids. Furthermore, the chain-length distribution of suberin monomers in primary ECWs ranged from C₁₆ to C₂₄, whereas in secondary and tertiary ECWs a shift towards higher chain lengths (C₁₆ to C₂₈) was observed. The lignin composition of Casparian strips (primary ECWs) showed a high syringyl content and was similar to lignin in secondary cell walls of the tertiary ECWs, whereas lignin in secondary ECWs contained higher amounts of p-hydroxyphenyl units. The suberin and lignin compositions of RHCWs rarely changed with increasing root age. However, compared to the suberin in ECWs, where C₁₆ and C₁₈ were the most prominent chain lengths, the suberin of RHCWs was dominated by the higher chain lengths (C₂₄ and C₂₆). The composition of RHCW lignin was similar to that of secondary-ECW lignin. Using lignin-specific antibodies, lignin epitopes were indeed found to be located in the Casparian strip. Surprisingly, the mature suberin layers of tertiary ECWs contained comparable amounts of lignin-like epitopes. Received: 19 August 1998 / Accepted: 3 February 1999     

Fusion and fission of plasma-membrane material accommodates for osmotically induced changes in the surface area of guard-cell protoplasts

Stomatal movement requires large and repetitive changes in cell volume and consequently changes in surface area. The patch-clamp technique was used to monitor changes in plasma-membrane surface area of individual guard-cell protoplasts (GCPs) by measuring membrane capacitance (C_m), a parameter proportional to the surface area. The membrane capacitance increased under hypoosmotic conditions and decreased after hypertonic treatment. As the specific capacitance remained constant, this demonstrates that osmotically induced changes in surface area are associated with incorporation and removal of membrane material. Osmotically induced fusion and fission of plasma-membrane material was not affected by removal of extracellular Ca²⁺. Dialysing protoplasts with very low (<2 nM) or high (1 μM) Ca²⁺ had no effect on changes in C_m under hypo- and hyperosmotic conditions. However, the rate of change in surface area was dependent on the size of the difference in osmotic potential applied. The larger the osmotic difference and thus changes in membrane tension caused by water influx or efflux, the faster the change in C_m. The results therefore demonstrate that osmotically induced fusion and fission of plasma-membrane material in GCPs are Ca²⁺-independent and modulated by membrane tension. Received: 10 February 1998 / Accepted: 21 April 1998     

THE DINOFLAGELLATE PELLICULAR WALL LAYER AND ITS OCCURRENCE IN THE DIVISION PYRRHOPHYTA1

Forty-five species of dinoflagellates were surveyed for the presence of a pellicular layer in the amphiesma or cell covering. Such a layer was found in 15 of the 20 genera studied. Half the pellicles tested were resistant to acetolysis and may contain a sporopollenin-like material similar to that of some dinoflagellate cyst walk. Most organisms which formed pellicles were capable of reinforcing this layer with cellulose. Pellicles of Heterocapsa niei (Loeblich) Morrill & Loeblich and Scrippsiella trochoidea (Stein) Loeblich were studied with the electron microscope. Evidence is presented indicating that dividing cells of S. trochoidea from new walls while still enclosed in the parental pellicular layer.     

Carbon isotopes and water use efficiency: sense and sensitivity

We revisit the relationship between plant water use efficiency and carbon isotope signatures (δ¹³C) of plant material. Based on the definitions of intrinsic, instantaneous and integrated water use efficiency, we discuss the implications for interpreting δ¹³C data from leaf to landscape levels, and across diurnal to decadal timescales. Previous studies have often applied a simplified, linear relationship between δ¹³C, ratios of intercellular to ambient CO₂ mole fraction (C _i/C _a), and water use efficiency. In contrast, photosynthetic ¹³C discrimination (Δ) is sensitive to the ratio of the chloroplast to ambient CO₂ mole fraction, C _c/C _a (rather than C _i/C _a) and, consequently, to mesophyll conductance. Because mesophyll conductance may differ between species and over time, it is not possible to determine C _c/C _a from the same gas exchange measurements as C _i/C _a. On the other hand, water use efficiency at the leaf level depends on evaporative demand, which does not directly affect Δ. Water use efficiency and Δ can thus vary independently, making it difficult to obtain trends in water use efficiency from δ¹³C data. As an alternative approach, we offer a model available at to explore how water use efficiency and ¹³C discrimination are related across leaf and canopy scales. The model provides a tool to investigate whether trends in Δ indicate changes in leaf functional traits and/or environmental conditions during leaf growth, and how they are associated with trends in plant water use efficiency. The model can be used, for example, to examine whether trends in δ¹³C signatures obtained from tree rings imply changes in tree water use efficiency in response to atmospheric CO₂ increase. This is crucial for predicting how plants may respond to future climate change. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Barrier function of the cell wall during uptake of nickel ions

Cell walls were isolated from roots of six plant species to study their ion-exchange capacity for nickel ions (S _Ni) at Ni²⁺ concentration of 10⁻³ M. The S _Ni values varied depending on the plant species from 50 to 150 μmol Ni²⁺ per gram dry wt; the sorption capacity increased in a row: Poaceae < Chenopodiaceae < Fabaceae. At pH 5 the sorption capacity of cell walls for nickel ions was determined by the presence of carboxyl groups of polygalacturonic acid in the polymeric cell-wall matrix. In all cases the ion-exchange capacity of cell walls was higher at pH 8 than at pH 5, indicating that Ni²⁺ binds also to a carboxyl group different from that of polygalacturonic acid. Irrespective of plant species, the presence of EDTA in the solution diminished drastically the absorption capacity of cell walls for Ni²⁺. It is concluded that the presence of 10⁻³ M EDTA weakens the defense properties of cell walls. The sequestration of Ni²⁺ in the cell wall can be considered as an effective means of plant cell defense against elevated concentrations of nickel ions in the external medium.     

An isotopic method for testing the influence of leaf litter quality on carbon fluxes during decomposition

During microbial breakdown of leaf litter a fraction of the C lost by the litter is not released to the atmosphere as CO₂ but remains in the soil as microbial byproducts. The amount of this fraction and the factors influencing its size are not yet clearly known. We performed a laboratory experiment to quantify the flow of C from decaying litter into the soil, by means of stable C isotopes, and tested its dependence on litter chemical properties. Three sets of ¹³C-depleted leaf litter (Liquidambar styraciflua L., Cercis canadensis L. and Pinus taeda L.) were incubated in the laboratory in jars containing ¹³C-enriched soil (i.e. formed C4 vegetation). Four jars containing soil only were used as a control. Litter chemical properties were measured using thermogravimetry (Tg) and pyrolysis–gas chromatography/mass spectrometry–combustion interface–isotope ratio mass spectrometry (Py–GC/MS–C–IRMS). The respiration rates and the δ¹³C of the respired CO₂ were measured at regular intervals. After 8 months of incubation, soils incubated with both L. styraciflua and C. canadensis showed a significant change in δ¹³C (δ¹³C_final = −20.2 ± 0.4‰ and −19.5 ± 0.5‰, respectively) with respect to the initial value (δ¹³C_initial = −17.7 ± 0.3‰); the same did not hold for soil incubated with P. taeda (δ¹³C_final:−18.1 ± 0.5‰). The percentages of litter-derived C in soil over the total C loss were not statistically different from one litter species to another. This suggests that there is no dependence of the percentage of C input into the soil (over the total C loss) on litter quality and that the fractional loss of leaf litter C is dependent only on the microbial assimilation efficiency. The percentage of litter-derived C in soil was estimated to be 13 ± 3% of total C loss.     

Antibacterial activity of ZnO nanoparticles prepared via non-hydrolytic solution route

The antibacterial activity of ZnO nanoparticles has been investigated and presented in this paper. Nanoparticles were prepared via non-hydrolytic solution process using zinc acetate di-hydrate (Zn(CH₃COO)₂·2H₂O) and aniline (C₆H₅NH₂) in 6 h refluxing at ∼65 °C. In the presence of four pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae, the antibacterial study of zinc oxide nanoparticles were observed. The antibacterial activity of ZnO nanoparticles (ZnO-NPs) were studied by spectroscopic method taking different concentrations (5–45 μg/ml) of ZnO-NPs. Our investigation reveals that the lowest concentration of ZnO-NPs solution inhibiting the growth of microbial strain is found to be 5 μg/ml for K. pneumoniae, whereas for E. coli, S. aureus, and S. typhimurium, it was calculated to be 15 μg/ml. The diameter of each ZnO-NPs lies between “20 and 30 nm” as observed from FESEM and transmission electron microscopy images. The composition of synthesized material was analyzed by the Fourier transform infrared spectroscopy, and it shows the band of ZnO at 441 cm⁻¹. Additionally, on the basis of morphological and chemical observations, the chemical reaction mechanism of ZnO-NPs was also proposed.     

Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves

Summary Protoplasts were isolated from oat (Avena sativa L.) leaves by the combination of highly purified preparations of pectin lyase, xylanase, and cellulase C₁. During the enzymic isolation, superoxide radical (O ₋ ² ) was generated from the tissues. Both the protoplasts themselves and the cell walls, exposed to enzyme treatment, produced O ₋ ² . Hydrogen peroxide (H₂O₂) apparently accumulated in the reaction mixture due to the spontaneous dismutation reaction of O ₋ ² , while a part of H₂O₂ may have been produced directly from cell walls by the action of enzymes. Singlet molecular oxygen (¹O₂) generated in the reaction mixture was detected by cholesterol oxidation in small unilamellar liposomes. It seems likely that¹O₂ may be generated by the peroxidase-H₂O₂-halide system during enzymic treatment of the leaves. The work was partially supported by the Research Project “Research and development of the improvement of bacterial and plant cells by cell fusion” of the Food and Agriculture Research and Development Association (Japan).     

Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus

 Callus induction, somatic embryogenesis and plant regeneration were obtained in six different citrus species [Citrus deliciosa Ten. (cv 'Avana'), C.limon (L.) Burm. (cv 'Berna'), C.madurensis Lour. (cv 'CNR P9'), C.medica L. (cv 'Cedro di Trabia'), C.tardiva Hort. ex Tan. (cv 'CNR P6'), C.sinensis (L.) Osb. (cv 'Ugdulena 7')] from cultures of pistil transverse thin cell layer explants [(t)TCL]. Explants were cultured on three different media: the nutrients and vitamins of Murashige and Skoog medium alone (MS) or MS supplemented with either 500 mg l^–1 malt extract (MS I) or 500 mg l^–1 malt extract and 13.3 μM 6-benzylaminopurine (MS II). Sucrose (146 mM) was used as the carbon source. Somatic embryos were visible 2–5 months after culture initiation. The different genotypes showed a different embryogenic frequency from stigma, style and ovary (t)TCL explants. All of the cultivars regenerated somatic embryos. Percentages of style (t)TCL explants producing somatic embryos ranged from 0% (C.deliciosa, C.madurensis, C.sinensis and C.tardiva on the three different media) to 5.2% (C.limon on MS II). Embryo formation in stigma (t)TCL explants ranged from 0% (C.madurensis on MS and MS I, C.sinensis on MS, C.deliciosa and C.tardiva on the three different media) to 42.4% (C.limon on MS II). Embryo formation in ovary (t)TCL explants ranged from 0% (C.deliciosa on MS, C.limon, C.medica, and C.sinensis on the three different media) to 9.3% (C.tardiva on MS I). After about 12 weeks somatic embryos developed into plantlets at a high frequency. Received: 22 September 1998 / Revision received: 6 November 1998 / Accepted: 23 November 1998     

Does Bienertia cycloptera with the single-cell system of C(4) photosynthesis exhibit a seasonal pattern of delta (13)C values in nature similar to co-existing C (4) Chenopodiaceae having the dual-cell (Kranz) system?

Family Chenopodiaceae is an intriguing lineage, having the largest number of C₄ species among dicots, including a number of anatomical variants of Kranz anatomy and three single-cell C₄ functioning species. In some previous studies, during the culture of Bienertia cycloptera Bunge ex Boiss., carbon isotope values (δ¹³C values) of leaves deviated from C₄ to C₃−C₄ intermediate type, raising questions as to its mode of photosynthesis during growth in natural environments. This species usually co-occurs with several Kranz type C₄ annuals. The development of B. cycloptera morphologically and δ¹³C values derived from plant samples (cotyledons, leaves, bracts, shoots) were analyzed over a complete growing season in a salt flat in north central Iran, along with eight Kranz type C₄ species and one C₃ species. For a number of species, plants were greenhouse-grown from seeds collected from the site, in order to examine leaf anatomy and C₄ biochemical subtype. Among the nine C₄ species, the cotyledons of B. cycloptera, and of the Suaeda spp. have the same respective forms of C₄ anatomy occurring in leaves, while cotyledons of members of tribe Caroxyloneae lack Kranz anatomy, which is reflected in the δ¹³C values found in plants grown in the natural habitat. The nine C₄ species had average seasonal δ¹³C values of −13.9‰ (with a range between species from −11.3 to −15.9‰). The measurements of δ¹³C values over a complete growing season show that B. cycloptera performs C₄ photosynthesis during its life cycle in nature, similar to Kranz type species, with a seasonal average δ¹³C value of −15.2‰. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of side-chain orientation on the <Superscript>13</Superscript>C chemical shifts of antiparallel β-sheet model peptides

The dependence of the ¹³C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel β-sheet model peptide represented by the amino acid sequence Ac-(Ala)₃-X-(Ala)₁₂-NH₂ where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel β-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the ¹³C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel β-sheet, there is (i) good agreement between computed and observed ¹³C^α and ¹³C^β chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed ¹³C^α and ¹³C^β chemical shifts as a function of χ¹ for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed ¹³C^α chemical shifts on χ^ξ (with ξ ≥ 2) compared to χ¹ for eleven out of seventeen residues. Our results suggest that predicted ¹³C^α and ¹³C^β chemical shifts, based only on backbone (φ,ψ) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Measuring <Superscript>13</Superscript>C<Superscript>β</Superscript> chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

A labeling scheme is introduced that facilitates the measurement of accurate ¹³C^β chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of ¹³C enrichment (30–40%) at C^β side-chain carbon positions for 15 of the amino acids with little ¹³C label at positions one bond removed (≈5%). A pair of samples are produced using [1-¹³C]-glucose/NaH¹²CO₃ or [2-¹³C]-glucose as carbon sources with isolated and enriched (>30%) ¹³C^β positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of ¹³C^β chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.     

The impact of elevated CO2 on growth and photosynthesis in Agrostis canina L. ssp. monteluccii adapted to contrasting atmospheric CO2 concentrations

 The aim of this study was to characterise growth and photosynthetic capacity in plants adapted to long-term contrasting atmospheric CO₂ concentrations (C _a). Seeds of Agrostis canina L. ssp. monteluccii were collected from a natural CO₂ transect in central-western Italy and plants grown in controlled environment chambers at both ambient and elevated CO₂ (350 and 700 μmol mol⁻¹) in nutrient-rich soil. Seasonal mean C _a at the source of the plant material ranged from 610 to 451 μmol CO₂ mol⁻¹, derived from C₄ leaf stable carbon isotope discrimination (δ¹³C). Under chamber conditions, CO₂ enrichment stimulated the growth of all populations. However, plants originating from elevated C _a exhibited higher initial relative growth rates (RGRs) irrespective of chamber CO₂ concentrations and a positive relationship was found between RGR and C _a at the seed source. Seed weight was positively correlated with C _a, but differences in seed weight were found to explain no more than 34% of the variation in RGRs at elevated CO₂. Longer-term experiments (over 98 days) on two populations originating from the extremes of the transect (451 and 610 μmol CO₂ mol⁻¹) indicated that differences in growth between populations were maintained when plants were grown at both 350 and 700 μmol CO₂ mol⁻¹. Analysis of leaf material revealed an increase in the cell wall fraction (CWF) in plants grown at elevated CO₂, with plants originating from high C _a exhibiting constitutively lower levels but a variable response in terms of the degree of lignification. In vivo gas exchange measurements revealed no significant differences in light and CO₂ saturated rates of photosynthesis and carboxylation efficiency between populations or with CO₂ treatment. Moreover, SDS-PAGE/ LISA quantification of leaf ribulose bisphosphate carboxylase/oxygenase (Rubisco) showed no difference in Rubisco content between populations or CO₂ treatments. These findings suggest that long-term adaptation to growth at elevated CO₂ may be associated with a potential for increased growth, but this does not appear to be linked with differences in the intrinsic capacity for photosynthesis. Received: 16 August 1996 / Accepted: 19 October 1996     

Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

SELF-STERILE AND MATURATION-DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA,CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

Homothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform-resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly-outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation-defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self-sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully-matured, chloroform-resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.     

Fourier transform infrared-spectroscopic characterisation of isolated endodermal cell walls from plant roots: chemical nature in relation to anatomical development

The chemical nature of enzymatically isolated endodermal cell walls from Cicer arietinum L., Clivia miniata Reg. and Iris germanica L. was studied by FTIR (Fourier transform infrared) spectroscopy. Observed frequencies were assigned to functional groups present in the cell wall and relative amounts of the biopolymers suberin and lignin, cell wall carbohydrates and proteins were determined. Infrared absorption spectra indicated structural characteristics for the three different developmental states of the isolated endodermal cell wall: primary endodermis with Casparian strips (state I), secondary endodermis with suberin lamellae (state II), and tertiary endodermis with U-shaped cell wall depositions (state III). The data obtained from this study are compared with previous results obtained by chemical degradation of isolated endodermal cell walls and subsequent determination of monomeric degradation products by gas chromatography and mass spectrometry. It is concluded that FTIR spectroscopy represents a direct and nondestructive method suitable for the rapid investigation of isolated plant cell walls. Furthermore, the observation that the suberin-assigned absorption bands disappeared after transesterification of the samples with BF₃-methanol confirmed that suberin is completely degraded by this treatment. Received: 20 February 1999 / Accepted: 25 May 1999     

FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950–1,185 cm⁻¹), protein (1,480–1,720 cm⁻¹), and the fatty acids (2,840–3,000 cm⁻¹) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as β-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.