Crystallographic structure of a helical lipopeptaibol antibiotic analogue

An X-ray diffraction analysis of the [Fmoc⁰,TOAC^4,8, Leu-OMe¹¹] analogue of thelipopeptaibol antibiotic trichogin A IV shows that the undecapeptide isfolded in a right-handed, mixed /3₁₀-helix. The helicalmolecules are connected in a head-to-tail arrangement along the b-axisthrough C=O···H-N intermolecular H-bonding. Thispacking mode generates a hydrophobic cavity where the FmocN-protecting groups are accommodated. The distances andangles between the nitroxide groups of the two TOAC residues, separated byone turn of the -helix, have been determined.     

5′-Isothiocyanato-N-1-naphthylphthalamic acid: a chemically reactive site-directed irreversible ligand for phytotropin receptors

A chemically reactive analog of the phytotropin N-1-naphthylphthalamic acid (NPA) was synthesized and evaluated as a site-directed irreversible ligand for the NPA receptor. The NPA analog (5-isothiocyanato-N-1-naphthylphthalamic acid; NCS-NPA) was synthesized in two steps. Pretreatment of etiolated Helianthus hypocotyl segments with NCS-NPA at concentrations in excess of 1 M resulted in a dose-dependent inhibition of basipetal [¹⁴C]IAA transport. Net uptake of IAA by hypocotyl segments was stimulated by NCS-NPA at concentrations of 1 M or greater. NCS-NPA inhibited the saturable binding of [³H]NPA in Helianthus microsomes in a dose-dependent fashion with 50% inhibition occurring at NCS-NPA concentrations of 3 to 10 nM. The binding affinity of [³H]NPA in microsomes pretreated with NCS-NPA followed by extensive washing was substantially reduced. These results demonstrate that NCS-NPA is a site-directed irreversible ligand for the NPA receptor and suggest that it may be of use in the purification and characterization of this biologically important receptor.Abbreviations ANPA 5-amino-naphthylphthalamic acid - IAA indole-3-acetic acid - NCS-NPA 5-isothiocyanato-N-1-naphthylphthalamic acid - NPA N-1-naphthylphthalamic acid - TLC thin-layer chromatography     

Growth stimulation ofEuphorbia lathyris L. by GA4+7 and BA

Container-grownEuphorbia lathyris plants were treated with foliar sprays of various combinations of BA and GA₄₊₇ or 0–3600 mg L^–1 Promalin (11 BA + GA₄₊₇) in separate experiments. GA₄₊₇ and Promalin stimulated plants to grow taller. BA and Promalin promoted axillary shoot growth. Multiple applications of Promalin stimulated branching more than single treatments. Dry weight accumulation was stimulated only if the growth regulators were applied to 28–33-cm and not to 56-cm tall plants. Chemical names used: (1, 2, 4a, 4b, 10)-2,4a,7-trihydroxy-1-methyl-8-methylenegibb-3-ene-1,10-dicarboxylic acid 1,4a-lactone (GA₄₊₇),N-(phenylmethyl)-H-purin-6-amine (BA), and Promalin [11 (wt/wt) GA₄₊₇ and BA].The use of the name Promalin or other trade names does not imply endorsement to the exclusion of other products or vendors that may also be suitable.     

Effects of G-protein probes on interactions between auxin efflux carriers and phytotropin receptors in zucchini (Cucurbita pepo L.) microsomal membranes

Microsomal vesicles prepared from etiolated hypocotyl tissue of zucchini (Cucurbita pepo L. cv. All Green Bush) exhibited saturable N-1-naphthylphthalamic acid ([³H]NPA) binding, NPA-stimulated association of indol-3yl-acetic acid ([³H]IAA), and saturable binding of guanosine 5-O-[3-thiotriphosphate] (GTP--[³⁵S]). These vesicles were used to test the possibility that NPA receptors might interact with IAA-anion efflux carriers by coupling through a GTP-binding protein (G-protein). Unlabelled GTP--S or guanosine 5-O-[2-thiodiphosphate] (GDP--S) had no effect on saturable NPA binding or on the NPA-stimulated association of IAA with microsomes. NPA did not affect saturable binding of GTP--[³⁵S] to microsomes, either in the presence or absence of saturating concentrations of unlabelled GTP--S or GDP. It is concluded that the occupancy of phytotropin receptors is not transduced to auxin efflux carriers by a GTP-binding protein.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Tetramethyl Rhodamine Methyl Ester (TMRM) is Suitable for Cytofluorometric Measurements of Mitochondrial Membrane Potential in Cells Treated with Digitonin

A new method for cytofluorometric analysis of mitochondrial membrane potential has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower and characterise the stability of . The method is suitable for sensitive measurement of in different types of cultured cells.     

Plant regeneration from callus and protoplast cultures of Lotus pedunculatus Cav.

Plant regeneration from leaf- and cotyledon-derived calli and from protoplast-derived tissue has been obtained in Lotus pedunculatus. Callus induction was achieved with 2,4-D and plant regeneration required the following two media sequences: bud formation was stimulated by IAA and BA and shoot growth by kinetin. Root formation occurred in the presence of IAA. Cotyledon protoplasts showed a low plating efficiency and plant regeneration was achieved via an intervening callus phase.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2iP N^6--2-isopentenyl-adenine - NAA -naphthaleneacetic acid     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1^-1BAP plus 2 mg 1^-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1^-1 did appear to determine the formation of bud-like embryos. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.     

Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) seedlings

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of -(14)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [¹⁴C]Gal from UDP-[¹⁴C]Gal into intact and partially hydrolyzed lupin -(14)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20–25°C in the presence of 25 mM Mn²⁺ and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M _r>150,000) from lupin we used was very low but increased when the M _r of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M _r (average M _r 60,000) -(14)-galactan was a more efficient acceptor [specific activity 2,000–3,000 pmol min^–1 (mg protein)^–1] than low-M _r (average M _r 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M _r galactan with endo--(14)-galactanase released mainly radioactive -(14)-galactobiose and Gal, indicating that the transfer of [¹⁴C]Gal occurred through -(14)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) -(14)-galactooligomers with degree of polymerization at least 5. Gal₇-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal_8–10-PA.Abbreviations AGP Arabinogalactan-protein - Ara Arabinose - DP Degree of polymerization - GalA Galacturonic acid - Gal _n -PA Pyridylaminated -(14)-galactooligosaccharides - GalT Galactosyltransferase - MALDI–TOF–MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - Rha Rhamnose Sugars described in this paper belong to the d-series unless otherwise noted     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Purification and properties of the α-3/4-l-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line

A soluble -3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700 000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminalN-acetylglicosamine residue in the Type 1 (Gal 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any -3-fucosyltransferase species acting solely onN-acetylglucosamine residues in Type 2 chains separated from the bulk of the -3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal -galactosyl residue and 2-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, andM _r, closely resembled those of the Lewis-blood-group gene associated -3/4-fucosyltransferase isolated from human milk.Deceased June 1991.     

The action of specific inhibitors of auxin transport on uptake of auxin and binding of N-1-naphthylphthalamic acid to a membrane site in maize coleoptiles

Using both 1-mm segments of corn (Zea mays L.) coleoptiles and a preparation of membranes isolated from the same source, we have compared the effectiveness of several inhibitors of geotropism and polar transport in stimulating uptake of auxin (indole-3-acetic acid, IAA) into the tissue and in competing with N-1-naphthylphthalamic acid (NPA) for a membrane-bound site. Low concentrations of 2,3,5-triiodobenzoic acid (TIBA), NPA, 2-chloro-9-hydroxyfluorene-9-carboxylic acid (morphactin), and fluorescein, eosin, and mercurochrome all stimulated net uptake of [³H]IAA by corn coleoptile tissues while higher concentrations reduced the uptake of both [³H]IAA and another lipophilic weak acid, [¹⁴C]benzoic acid. Since low concentrations of fluorescein and its derivatives competed for the same membrane-bound site in vitro as did morphactin and NPA, the basis for both the specific stimulation of auxin accumulation and the inhibition of polar auxin transport by all these compounds may be their ability to interfere with the carrier-mediated efflux of auxin anions from cells. At higher concentrations, the decrease in accumulation of weak acids was nonspecific and thus may be the result of acidification of the cytoplasm and a general decrease in the driving force for uptake of the weak acids. Triiodobenzoic acid was an exception. Low concentration of TIBA (0.1–1 M) were much less effective than NPA in competing for the NPA receptor in vitro, but little different from NPA in ability to stimulate auxin uptake. One possibility is that TIBA, a substance which is polarly transported, may compete with auxin for the polar transport site while NPA, morphactin, and the fluorescein derivatives may render this site inactive.Abbreviations C1-NPA 2,3,4,5-tetrachloro-N-1-naphthylphthalamic acid - IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthalenacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Turgor-dependent efflux of assimilates from coats of developing seed of Phaseolus vulgaris L.: water relations of the cells involved in efflux

The turgor-homeostat model of assimilate efflux from coats of developing seed of Phaseolus vulgaris L. was further characterised. The turgor pressure (P), the volumetric elastic modulus () and hydraulic conductivity (L_p) of the seed coat cells responsible for assimilate efflux and cotyledon storage parenchyma cells were determined with a pressure probe. In addition, turgor of the seed coat and cotyledons was estimated by measuring the osmolalities of symplastic and apoplastic fluids extracted by centrifugation. Osmolality of symplastic and apoplastic saps collected from the seed coat declined significantly over the period of seed development from a cotyledon water content of 80% to 50%. However, the difference in osmolalities of the apoplastic and symplastic saps remained relatively constant. For cotyledons, osmolality of the apoplastic sap exhibited a significant decline during seed development, while the osmolality of symplastic sap did not change significantly. Hence cotyledon P increased as the water content dropped from 80% to 50%. For both detached and attached empty seed coats, a small decrease (ca. 40mOsmol·kg^–1) in the osmolality of the bathing solution, led to a rapid increase in P of cells involved in assimilate efflux (efflux cells) by about 0.07 MPa. Thereafter, cell P exhibited a rapid decline to the original value within some 20–30 min. When P of the efflux cells was reduced by increasing the osmolality of the bathing solution, P exhibited a comparable rate of recovery for attached empty seed coats but there was no P recovery to its original value in the case of detached seed coats. In contrast, the cotyledon storage parenchyma cells did not exhibit P regulation when the osmolality of the bathing solution was changed. The observations that the efflux cells of P. vulgaris seed coats can rapidly adjust their P homeostatically in response to small changes in apoplastic osmolality are consistent with the operation of a turgor-homeostat mechanism. The volumetric elastic modulus () of the seed coat efflux cells exhibited a mean value of 7.3±0.8 MPa at P=0.15 MPa and was found to be linearly dependent on cell P. The e of the cotyledon storage parenchyma cells was estimated to be 6.1±1.0 MPa at P=0.41 MPa. Hydraulic conductivity (L_p) of the seed coat cells and the cotyledon cells was (8.2±1.5) × 10^–8m·s^–1·MPa^–1and (12.8±1.0) × 10^–8 m·s^–1·MPa^–1, respectively. The relatively high , i.e., low elasticity, for the seed coat cell walls would ensure that small changes in water potential of the seed apoplast will be reflected in large changes in cell P. The high L_p values for both the seed coat and the cotyledon cells is consistent with the rapid changes in P in response to changes in water potential of the seed apoplast.Abbreviations LYCH Lucifer Yellow CH - volumetric elastic modulus - L_p hydraulic conductivity - P turgor pressure - osmotic pressure - t_1/2 half-time for water exchange The investigation was supported by funds from the Australian Research Council. We are grateful to Louise Hetherington for competent technical assistance and to Kevin Stokes for raising the plant material.     

Inorganic carbon assimilation in the Isoetids,Isoetes lacustris L. and Lobelia dortmanna L.

Summary The inorganic carbon fixation patterns of Isoetes lacustris and Lobelia dortmanna from an oligotrophic Scottish loch have been examined by following titratable acidity changes in plant sap and light/dark ¹⁴CO₂ incorporation by roots and shoots. The diurnal pattern of titratable acidity changes in I. lacustris suggests crassulacean acid metabolism (CAM) while the lack of any change in titratable acidity in L. dortmanna suggests C₃ metabolism. Of the carbon fixed by L. dortmanna, 99.9% was taken up through the roots and fixation occurred primarily during the day. In Isoetes, CO₂ was taken up by both roots and shoots and during both day and night. Regardless of the site of CO₂ uptake, fixation occurred only in the shoots of both plants. Analysis of carbon isotope ratios of plant organic material was used to further investigate the photosynthetic mechanisms of these Isoetids. Considering the absence of a nighttime peak in titratable acidity in L. dortmanna, the ¹³C (=¹³C plant-¹³C source) value of the shoots of L. dortmanna (-14.2) is indicative of C₃ photosynthesis limited by the rate of CO₂ diffusion. The less negative of I. lacustris (-6.0) is consistent with both dark acidification of CAM and CO₂ limited C₃ photosynthesis. This is in contrast to the terrestrial Isoetes durieui which is shown to have a value which is similar to a terrestrial C₃ plant. The carbon fixation patterns of these Isoetids suggest that the CO₂ concentration in the loch may be growth limiting, and that root uptake and/or dark acidification are means of optimising CO₂ supply. However, in view of the relatively high levels of CO₂ in sediment and bulk water, it is suggested that low levels of nutrients may also limit growth in these plants.     

Effect of the support matrix on biotransformation of benzaldehyde to benzyl alcohol by yeast cells in aqueous and aqueous-organic two phase systems

Summary Biotransformation of benzaldehyde to benzyl alcohol bySaccharomyces cerevisiae immobilized in different support matrices was investigated. Polymers with intrinsic hydrophobic and/or hydrophilic nature as well as mixed hydrophobic and hydrophilic supports were examined both in aqueous and bisphasic aqueous-organic systems. The hydrophobic support material ENTP-2000 or mixed silicone:alginate (50-2550-75) proved to be most suitable not only for nonconventional media but also for conventional aqueous media for production of benzyl alcohol. With ENTP-2000, catalytic activity and maximum yield were 159 mol h^–1 g^–1 dry weight catalyst and 0.89 mM, respectively, in hexane containing 2% moisture. Corresponding values in aqueous media were 246 mol h^–1 g^–1 dry weight catalyst and 1.53 mM. With 5050 silicone:alginate, catalytic activity and maximum yield were 177 mol h^–1 g^–1 dry weight catalyst and 1.18 mM, respectively, in hexane containing 2% moisture. Corresponding values in aqueous media were 192 mol h^–1 g^–1 dry weight catalyst and 0.8 mM.     

Embryo development in reciprocal crosses of Phaseolus vulgaris L. and P. coccineus Lam.

Summary Embryo development was examined in reciprocal crosses of Phaseolus vulgaris cv. Great Northern and P. coccineus cv. Scarlet Runner. The formation of abnormal (shrunken and underdeveloped) embryos constituted the primary crossing barrier between the two species when P. coccineus was the female parent. Plants of P. coccineus X P. vulgaris were obtained by embryo culture. Although the P. vulgaris X P. coccineus cross resulted in normal seed development, the fertility of the resulting hybrids was much lower (27%) than that of the reciprocal hybrids (81%). Three classes of F₂ embryos, normal, shrunken, and underdeveloped were formed on reciprocal F₁s and the frequencies did not differ between reciprocal populations. Thus, the interactions between embryo and endosperm and/or maternal parent rather than cytoplasmic-nuclear effects seem to be important in the determination of the extent of embryo growth. The examination of pollen fertility of F₂ plants and the development of F₂ and F₃ embryos suggests that the formation of abnormal embryos and reduced male fertility are independent events. The P. vulgaris — P. coccineus crosses may be useful in studying the possible involvement of interspecific differences in hormonal metabolism in the development of hybrid embryos.     

Structural and Functional Rearrangements of Mesophyll as a Probable Basis for the Cytokinin-Dependent Assimilate Translocation in Detached Leaves

The effects of benzyladenine (BA) on the mesophyll functioning, such as osmotic potential (), the effect of the inhibitors of ⁺-ATPase on the influx of ¹⁴C-sucrose, the direction of carbon metabolism, and the rate of dark respiration, were followed in the detached leaves of pumpkin (Cucurbita pepo L.) and broad beans (Vicia faba L.). BA elevated and established a gradient of (_p) between the treated and untreated leaf regions. The inhibitors of H⁺-ATPase did not affect the BA-induced influx of ¹⁴C-sucrose. The changes were accompanied with the elevated synthesis of starch and other polymeric compounds and the diminished synthesis of the substances of relatively low molecular weight. The stimulation of dark respiration was short and inconsiderable. The author concludes that the BA-induced transport was a passive process related to a increase. Leaf expansion accompanied by the synthesis of high-molecular-weight substances essential for cell growth and by starch synthesis apparently increased the sink capacity of the BA-treated detached leaves. The diminished efflux from the leaf blade was probably related to a lowered level of the transportable carbon compounds restricting their entry into the phloem. The influx induction could result from the activation of growth and metabolic processes, the decline in the number of organic molecules per cell volume unit, and the development of _p between the source and sink leaf regions.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Plant regeneration from isolated microspores of linseed (Linum usitatissimum L.)

Embryogenesis and plant regeneration were induced in isolated microspore culture of linseed (oilflax, Linum usitatissimum). Microspores underwent cell divison which led to either microcallus or embryoid formation, when they were cultured in a modified liquid Nitsch-Lichter-Nitsch medium (Lichter 1985) at two different incubation temperatures (30 and 35 °C); some embryoids and microcalli further developed to larger calli. After transfer of the microspore derived calli to a solid medium containing zeatin (Img 1^–1) shoot induction was achieved from 36 to 66% of the calli. The highest frequency of regenerated plants was obtained in microspore cultures of the hybrid Atalante x Szegedi 62 (F₁) at 30 °C, whereas for the second genotype Pedigree 2 x Kiszombori 41 (F₂) the higher incubation temperature seemed to be more efficient. Shoots could be successfully rooted on an indole acetic acid containing medium and then transplanted to vermiculite and finally to soil. Most of the plants survived the transfer into soil in the greenhouse, where they could be successfully grown to maturity.Abbreviations BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - IAA indole acetic acid - N6 Chu (1978) medium - NAA naphthaleneacetic acid - NLN Nitsch-Lichter-Nitsch (1985) - MS Murashige and Skoog (1962) medium - ZEA zeatin