Electrical field effects induced in membranes of developing chloroplasts

Etioplasts, etiochloroplasts, and chloroplasts of Avena sativa L. purified on a Percoll gradient were subjected to increasing electric field strengths in the orifice of a hydrodynamically focussing Coulter Counter. The change in resistance of the orifice when an organelle is present correlates well with the size of the plastid for field strengths up to about 3.5 kV cm^-1. Beyond this field strength, depending on the size of the organelle, the size is underestimated. The underestimation of the size is caused by the dielectric breakdown of the envelope membranes once a critical membrane potential has been exceeded. Beyond breakdown the signal of the particle is predominately determined both by the internal conductivity and the increased membrane conductivity. Measurements of the breakdown voltage of different developmental stages of the plastids reveal that the breakdown voltage decreases from 1.2 V in etioplasts to about 0.9 V in chloroplasts after 48 h illumination. The decrease in breakdown voltage can be explained in terms of increasing incorporation of proteins into the inner envelope membrane during development.This view is consistent with conclusions drawn by other authors from transport and biochemical studies. The underestimation of the size beyond breakdown is about 20% and increases to a constant value of about 40% during the first 3 h of illumination. The underestimation decreases again to about 10% when the chloroplast stage is reached. This result is consistent with the current view of chloroplast development. Mobilisation of glucans, the transformation of the prolamellar body of etioplasts into thylacoid membranes as well as an intensive synthesis of pigments and enhanced rates of ions transport in the first hour of illumination gives rise to an increased pool of ionic compounds within the plastid stroma.It should be noted that purification of the plastids on Percoll gradient leads to size distributions which are almost normally distributed over the whole field range, suggesting that the preparations are also electrically homogeneous (U. Zimmermann, F. Riemann and G. Pilwat: Biochim. Biophys. Acta 436, 460–474 (1976)). In contrast with results of Lürssen, K., Z. Naturforsch. 25b, 1113–1119 (1970) only a slight increase of the modal volume from the etioplast stage to the chloroplast stage is observed.     

Integral association of phytochrome with a membranous fraction from etiolatedAvena shoots: red/far-red photoreversibility and in vitro characterization

The results reported in this paper provide strong evidence to support the belief that the small percentage of phytochrome recovered in low-speed centrifugation pellets, when prepared in the absence of divalent cations after various in vivo irradiations, is not simply a manifestation of non-specific co-precipitation of soluble phytochrome.The far-red reversibility of the observed near-doubling of phytochrome pelletability after in vivo red irradiation indicates that phytochrome pelletability in the absence of divalent cations is a phytochrome-controlled response. The characteristics of the pelleted phytochrome indicate a strong, hydrophobic interaction with membranes. A tentative proposal to explain the observed characteristics of the association of phytochrome with membranous material in the absence of divalent cations after different in vivo irradiations has been put forward.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the fat-red light absorbing form - Ptot total phytochrome - R red light irradiation - FR far-red light irradiation     

Overcoming dormancy in seeds with ethanol and other anesthetics

Dormancy in fall panicum (Panicum dichotomiflorum Michx.) caryopses (seeds) is overcome by imbibition at 35° C in ethanol solutions. Whereas germination in the absence of ethanol depends on active phytochrome, the seeds may germinate in darkness after treatment with 0.2 to 0.5 M ethanol. Ethanol overcomes dormancy also in seeds of several other weedy grass species. Ethyl ether, chloroform, methanol, and acetone act similarly to ethanol. We suggest that this action depends on modifyng the properties of a membrane(s) in a manner related to the actions of other anesthetics.     

The stability of the postulated wall-loosening enzyme in acid-induced growth

Long-term pretreatments with cycloheximide (CH) caused inhibition of subsequent acidinduced growth of Avena coleoptile segments, but only after 6 or more h of CH treatment. These results together with previous, published evidence with frozen-thawed tissue are consistent with the hypothesis that there exists a wall-loosening enzyme responsible for acid-induced elongation and that it has a half-life of at least 7–8 h.     

The use and misuse of calcium carbonate as an aid to the spectrophotometric assay of phytochrome in vitro

Supernatant and resuspended pellet samples from a centrifugation of homogenised, etiolated oat seedlings were prepared and assayed spectrophotometrically for phytochrome in the presence and absence of added calcium carbonate (CaCO₃) particles under a variety of conditions. At a constant sample thickness, in the absence of CaCO₃, increasing sample concentration had no significant effect on the expected phytochrome reading. In the presence of CaCO₃, however, as sample concentration increased, the phytochrome reading was less than, expected more so in resuspended pellet samples than in supernatant samples. At a constant sample concentration in the absence of CaCO₃, increasing sample thickness gave no significant difference from the excepted phytochrome reading in supernatant samples, but led to a slight increase over the expected phytochrome reading in resuspended pellet samples. In the presence of CaCO₃, increasing sample thickness led to a drop from the expected phytochrome reading in both sample types, but more so in resuspended pellet samples. These findings show that the use of CaCO₃ as an aid to spectrophotometric phytochrome assay can lead to large artifacts in the instrument reading and that its use should be approached with caution.     

The stromacentre inAvena plastids: an aggregation ofβ-glucosidase responsible for the activation of oat-leaf saponins

The stromacentre, a particular structure in the plastids of mostAvena species, was isolated from etioplasts ofAvena sativa and then characterized to determine its biological function. When comparing differentAvena species with or without stromacentre, it was shown that the stromacentre, a 63-kDa protein, and saponins (characteristic compounds ofAvena sativa) either occur together or not at all. This linkage was confirmed by demonstrating a transformation of saponins by the isolated stromacentre protein: avenacosides were hydrolyzed to 26-desgluco-avenacosides. Therefore, the stromacentre protein had to be regarded as a-glucosidase. Enzyme assays usingp-nitrophenyl--d-glucopyranoside as substrate showed that this-glucosidase has a pH optimum at pH 6.0. The calculatedK _m value for this substrate was 2.2·10^-3 M. Antibodies against the stromacentre protein inhibited-glucosidase activity. The determination of the molecular weight of the-glucosidase by sodium dodecyl sulfate-gel electrophoresis showed that it consists of subunits of 63 kDa. After gel electrophoresis under non-denaturing conditions, enzymatically active molecules were shown to consist of at least two of these subunits. Molecules aggregated up to about 10⁶ Da also had enzyme activity. Enzyme assays using avenacosides as substrate showed a pH optimum at pH 6.0. The calculatedK _m value for this substrate was 1.2·10^-5 M. The high affinity to the avenacosides and the high specificity for the C-26 bound glucose indicate that avenacosides are the natural substrates for this-glucosidase. Assuming that the avenacosides in oat leaves play a role as preformed chemical inhibitory substances against phytopathogenic microorganisms, a model is presented showing the stromacentre with a central role in activating the fungitoxicity of avenacosides.     

The role of the apex in the phototropic curvature of Avena coleoptiles: positive curvature under conditions of continuous illumination

The differential growth causing second positive phototropic curvature in intact, black-capped and decapitated Avena coleoptiles has been measured. In all cases the curvature is brought about by a cessation in growth of the illuminated side. The fact that shading the apex does not significantly alter the initial steps of differential growth means that the subapical zones can perceive and respond to unilateral illumination. Decapitation significantly reduces coleoptile growth, especially in the most apical zone. However, the fact that differential growth is still evident in the other zones of decapitated coleoptiles within 30 min of unilateral illumination requires one to conclude that the apex cannot be controlling the differential growth in those basal zones.     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Reversal by pressure of seed germination promoted by anesthetics

Germination of Panicum capillare L. caryopses induced by solutions of ethanol and ethyl ether was prevented by application of pressure >1 MPa during the period of exposure to the anesthetic. This effect of pressure indicates that germination is correlated with expansion at a site of anesthetic action in a cell membrane. The effects of several other anesthetics were measured on germination of P. capillare seeds. Ethanol, chloroform, and ethyl ether had the highest activity. Methanol and isopropanol were inactive. The effective compounds are thought to distribute preferentially to lipid-solution interfaces in cell membranes of the seeds.     

Wintertime changes in the ultrastructure and metabolism of the microsporangiate strobili of the Scotch pine

Terminal buds of Pinus silvestris L. containing microsporangiate strobilus primordia were collected once a month throughout the winter. The electron microscopic studies indicated that in October and December, the cells of the strobili contained a large number of vacuoles, a portion of which was supposedly autophagic, and stacked rough endoplasmic reticulum. By February, the amount of these had decreased, and instead, a large population of dense bodies was visible. Additional phenomena, characteristic at this state, were the occurrences of highly uneven contours of the plasmalemma and of inclusions of various kinds between the plasmalemma and the cell wall. In March, autolysis was visible in a portion of cells outside the sporangia. In the sporangia the ground cytoplasm was thin but the number of organelles was increasing. In the April collections, cell divisions were visible. The amount of protein per dry weight increased during the winter reaching a peak in February. The activity of RNases, having optima of pH 5.0 and pH 7.5, was measured in two successive years. Both series showed a period of high activity during the middle of the winter. The exact timing of this period depended on the year in question. On the basis of these observations, the dormant period of the microsporangiate strobili of the Seotch pine is divided into three sub-periods. It is also suggested that the definition of dormancy of these structures should include a mentioning of alterations in the metabolical machinery of the cells.Abbreviations CH chromosome - CW cell wall - D dictyosome - ER endoplasmic reticulum - L lipid spherule - M mitochondrion - N nucleus - NE nuclear envelope - P plasma membrane - Pp proplastid - RER rough ER - V vacuole     

Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolatedAvena sativa L.: Conclusions on the conformation of the native protein

Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543–8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.Abbreviations kDa kilodalton - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor W. Rau on the occasion of his 60th birthday.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species

Seven monoclonal antibodies (MAbs) have been prepared to phytochrome from green oat (Avena sativa L. cv. Garry) leaves. One of these MAbs (GO-1) cross-reacts with apoprotein of the phytochrome that is most abundant in etiolated oat shoots as assessed by immunoblot assay of fusion proteins expressed in Escherichia coli. The epitope for this MAb is located between amino acids 618 and 686 in the primary sequence of type 3 phytochrome (Hershey et al. 1985, Nucleic Acids Res. 13, 8543–8559), which is one of the predominant phytochromes in etiolated oats. Three other MAbs (GO-4, GO-5, GO-6) immunoprecipitate phytochrome isolated from green oat leaves, as evaluated by photoreversibility assay. GO-1, GO-4, GO-5 and GO-6 are therefore directed to phytochrome. While evidence obtained with the other three MAbs (GO-2, GO-7, GO-8) strongly indicates that they are also directed to phytochrome, this evidence is not as rigorous. Recognition of antigen by any of these seven MAbs is not significantly reduced by periodate oxidation, indicating that their epitopes probably do not include carbohydrate. All but GO-1 bind either very poorly or not at all the phytochrome that is abundant in etiolated oat shoots. These data reinforce earlier observations made with antibodies directed to phytochrome from etiolated oats, indicating (1) that the phytochromes that predominate in etiolated and green oats differ immunochemically and (2) that phytochrome preparations from green oat leaves contain very little of the phytochrome that is abundant in etiolated shoots. An hypothesis that these two immunochemically distinct phytochromes form heterodimers in vitroAbbreviations Da Dalton - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - HA hydroxyapatite - Ig immunoglobulin - MAb monoclonal antibody - SDS sodium dodecyl sulfate is supported by comparison of immunoblot data obtained with conventionally purified phytochrome from etiolated oats to that expressed as fusion protein in E. coli.This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Lyle Crossland and Ms. Sue Kadwell for their assistance in the construction of the cDNA clones, and Dr. Gyorgy Bisztray for providing us with clone pCBP3712. Dr. Phillip Evans and Dr. Russell Malmberg kindly provided MAbs 4F3, 6F12 and 8C10, as well as a corresponding antigen preparation. The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.