Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonio lyase Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged (G.W., We 630/9-7; We 630/10-1). Thanks are given to Dr. St. Kellam (Department of Plant Microbiological Sciences, University of Canterbury, New Zealand) for correcting the English.     

A comparative study into the chemical constitution of cutins and suberins from Picea abies (L.) Karst., Quercus robur L., and Fagus sylvatica L.

The compositions of BF₃/CH₃OH depolymerisates of cutins and suberins from leaf and periderm samples from Picea abies [L.] Karst., Quercus robur L., and Fagus sylvatica L., respectively, were determined by quantitative capillary gas chromatography/mass spectroscopy. Long-chain monobasic, -hydroxymonobasic, dihydroxymonobasic, trihydroxymonobasic and epoxyhydroxymonobasic alkanoic acids constituted the major aliphatic monomers of leaf cutins. The total amounts of cutin monomers ranged from 629 mg · m^–2 (Fagus) to 1350 mg · m^–2 (Quercus). Cutin composition and amounts did not significantly differ between current year and three-year-old needles of Picea. Trans-esterification of periderm samples yielded a much greater variety of aliphatic monomers than obtained from cutins. In addition to the substance classes found with cutins, suberin depolymerisates also contained , -dibasic acids while dihydroxymonobasic acids were lacking. Depolymerisates from periderms taken from different locations on a Picea tree did not differ significantly in their relative composition. The results are discussed in terms of the distinctive characteristics of the aliphatic portions of cutins and suberins, respectively. Discriminant analysis is applied for formulating a quantitative and inarbitrary classification rule for cutins and suberins. The precision, statistical significance and robustness of this classification rule are tested by employing it to a large set of compositional data (70 plant species) from the literature. The relevance of data obtained by depolymerization methods for elucidating the physical structure of cutins and suberins in situ is evaluated.To whom correspondence should be addressedThe authors are indebted to Drs. J. Winkler and H. Krause (Laboratorium für Strukturchemie des Fachbereichs Chemie, Biologie und Geowissenschaften, Technische Universität München, Garching, FRG) for performing capillary gas chromatography-mass spectrometry and their valuable help in the identification of cutin and suberin constituents. The work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bayerisches Staatsministerium für Unterricht, Kultus, Wissenschaft und Kunst.     

Floral morphogenesis in Anagallis: Scanning-electron-micrograph sequences from individual growing meristems before,during, and after the transition to flowering

Non-destructive scanning electron microscopy allows one to visualize changing patterns of individual cells during epidermal development in single meristems. Cell growth and division can be followed in parallel with morphogenesis. The method is applied here to the shoot apex of Anagallis arvensis L. before, during, and after floral transition. Phyllotaxis is decussate; photoperiodic induction of the plant leads to the production of a flower in the axil of each leaf. As seen from above, the recently formed oval vegetative dome is bounded on its slightly longer sides by creases of adjacent leaf bases. The rounded ends of the dome are bounded by connecting tissue, horizontal bands of node cells between the opposed leaf bases. The major growth axis runs parallel to the leaf bases. While slow-growing at the dome center, this axis extends at its periphery to form a new leaf above each band of connecting tissue. Connecting tissue then forms between the new leaves and a new dome is defined at 90° to the former. The growth axis then changes by 90°. This is the vegetative cycle. The first observed departure from vegetative growth is that the connecting tissue becomes longer relative to the leaf creases. Presumably because of this, the major growth axis does not change in the usual way. Extension on the dome continues between the older leaves until the axis typically buckles a second time, on each side, to form a second crease parallel to the new leaf-base crease. The tissue between these two creases becomes the flower primordium. The second crease also delimits the side of a new apical dome with the major axis and growth direction altered by 90°. During this inflorescence cycle the connecting tissue is relatively longer than before. Much activity is common to both cycles. It is concluded that the complex geometrical features of the inflorescence cycle may result from a change in a biophysical boundary condition involving dome geometry, rather than a comprehensive revision of apical morphogenesis.Abbreviation SEM scanning electron microscopy, micrograph Use of the SEM facility of Professor G. Goffinet, Institute of Zoology, University of Liège, is greatly appreciated. We thank Dr. R. Jacques, C.N.R.S., Le Phytotron, Gif-sur-Yvette, France, for providing the experimental material, and Mr. Philippe Ongena for expert photography. Support was from grants from the U.S. Department of Agriculture and National Science Foundation as well as from the Fonds National de la Recherche Scientifique, Fonds de la Recherche Fondamentale et Collective, and the Action de Recherche Concertée of Belgium.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

Mechanism for formation of the secondary wall thickening in tracheary elements: Microtubules and microfibrils of tracheary elements of Pisum sativum L. and Commelina communis L. and the effects of amiprophosmethyl

Arrangements of microfibrils (MFs) and microtubules (MTs) were examined in tracheary elements (TEs) of Pisum sativum L. and Commelina communis L. by production of replicas of cryo-sections, and by immunofluorescence microscopy, respectively. The secondary wall thickenings of TEs of Pisum and Commelina roots have pitted and latticed patterns, respectively. Most MFs in the pitted thickening of Pisum TEs retain a parallel alignment as they pass around the periphery of pits. However, some groups of MFs grow into the pits but then terminate at the edge of the thickening, indicating that cellulose-synthase complexes are inactivated in the plasma membrane under the pit. Microtubules of TEs of both Pisum and Commelina are localized under the secondary thickening and few MTs are detected in the areas between wall thickenings. In the presence of the MT-disrupting agent, amiprophosmethyl, cellulose and hemicellulose, which is specific to secondary thickening, are deposited in deformed patterns in TEs of Pisum roots, Pisum epicotyls and Commelina roots. This indicates that the localized deposition of hemicellulose as well as cellulose involves MTs. The deformed, but heterogeneous pattern of secondary thickening is still visible, indicating that MTs are involved in determining and maintaining the regular patterns of the secondary thickening but not the spatial heterogeneous pattern of the wall deposition. A working hypothesis for the formation of the secondary thickening is proposed.Abbreviations APM amiprophosmethyl - DMSO dimethyl sulfoxide - F-WGA fluorescein-conjugated wheat-germ agglutinin - M F microfibril - MT microtubule - PEG polyethyleneglycol - TE tracheary element I thank Ms. Aiko Hirata (Institute of Applied Microbiology, University of Tokyo, Japan) for help in taking stereomicrographs. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.     

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase,but not ferredoxin-NADP reductase,controls electron flow to phosphoglycerate in the dark-light transition

P₇₀₀ is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P₇₀₀ is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)^–1 electrons in geranium leaves, 16 nmol · (mg chlorophyll)^–1 in sunflower and 22 nmol · (mg chlorophyll)^–1 in oleander. Reduction of NADP during the initial phase of P₇₀₀ oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.Abbreviations Chl chlorophyll - P₇₀₀ electron donor pigment in the reaction center of photosystem I Cooperation of the Institute of Botany of the University of Würzburg with the Institute of Astrophysics and Atmospheric Physics of the Estonian Academy of Sciences in Tartu was supported by the Deutsche Forschungsgemeinschaft and the Estonian Academy of Sciences. This work was performed within the Sonderforschungsbereich 251 of the University of Würzburg.