Johannes Willenbrink

Accumulation of sucrose in vacuoles isolated from red beet tissue

Vacuoles were isolated from red beets (Beta vulgaris L.) by slicing the tissue and separated using a discontinuous dextran gradient centrifugation. The uptake of sucrose against a concentration gradient into the dextran-impermeable [3H]-H2O space of these organelles was studied using silicone layer filtering centrifugation on both fluorometric and 14C-measurement of sucrose. The rate is 24 nmol sucrose (unit betacyanin)-1 h-1 and appears to be stimulated by ATP to an uptake rate of 34 nmol. Control experiments with slices cut from red beet tissue and incubated with [14C]sucrose gave comparable results. An ATPase activity dependent on both Mg2+ and K+ seems to be localized at the inner surface of the tonoplast. This activity is strongly inhibited by EDAC and tartrate and there is no effect of oligomycin, whereas a slight stimulation was caused by DCCD.

Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissue. Kinetics and specifity of the sucrose uptake system

The uptake of sucrose against a concentration gradient into the dextran-impermeable [3H]H2O space of red beet (Beta vulgaris L.) vacuoles has been studied using silicone-layer-filtering centrifugation on both fluorometric and 14C-measurement of sucrose. Sucrose transport into vacuoles proceeds partly by an active transport system and partly by passive permeation. The KM(20°C) for active sucrose uptake was found to be about 22 mM and the VMax(20°C) was about 174 nmol sucrose x (unit betacyanin)-1 x h-1. The temperature dependency of sucrose transport appears to have an activation energy of 35,0 KJ×mol-1. Among various mono-, di-, and trisaccharides tested, raffinose acts as a competitive inhibitor of sucrose uptake.

CO2-fixierung und stofftransport in benthischen marinen algen. II. zum ferntransport 14C-markierter assimilate bei laminaria hyperborea und laminaria saccharina

Summary 1. Transport of 14 C-labelled assimilates was studied during the main growth period of two Laminaria species. H14CO3- was fed to the plants using a plexiglas chamber tightly fixed to the phylloid 40–50 cm from the growing region. The phylloids were harvested after 3–96 hours and cut into segments 15 mm in length. Radioactivity was determined by dry combustion and subsequent liquid scintillation counting. Labelled sugars, amino acids and organic acids were analysed after extraction with 80 % and 50 % ethanol by chromatography. The pathway of translocation was studied by histoautoradiography. 2. Nearly all of the labelled translocate moved towards the base of the phylloid, and significant accumulation of translocate occurred within the growing region of the phylloid and in growing haptera. 3. The labelled translocate consisted of mannitol (53 %), amino acids (alanine, glycine, serine, glutamic acid and some others; 45 %) and malate (2 %). This composition is similar to that of the products of photosynthesis in L. saccharina . 4. In most experiments the translocation velocity was about 5 cm/h, and the maximum velocity did not exceed 10 cm/h. 5. Certain groups of cells within the medulla of the phylloid were identified as the conducting tissue.

Die pflanzliche Vakuole als Speicher

Plant cell vacuoles not only maintain cellular turgidity, but are known as a deposit for both lytic enzymes and end products noxious for the cytoplasm. In addition, vacuoles accumulate nutritive solutes and function as a storage pool for proteins, sugars, organic acids etc. Long-term as well as diurnal storage and release processes emphasize the role of the central vacuole in metabolic regulation in a plant cell. D ie groBe, dem Mikroskopiker wohlvertraute Zentralvakuole der Pflanzenzelle ist als Teil des funktionell hochdifferenzierten Endomembransystems eine vergleichsweise volumin6se Komponente, die sich im Laufe der Zelldifferenzierung erst als Folge eines st/indigen Membranflusses aus dem ER-System fiber den Golgi-Apparat herausbildet. Sowohl der Innenraum der Vakuole als auch die vom Endomembransystem insgesamt umschlossenen Rfiume, ferner die Kernhfille, die R/iume zwischen innerer und /iuBerer Mitochondrienund Chloroplastenmembran sowie endlich das Thylakoidkompartiment werden der Matrixphase als sog. Vakuom gegeniibergestellt. Matrixphase und Vakuom sind gegeneinander durch Membranen lfickenlos abgegrenzt. Physikalische und chemische Eigenschaften der Membranen entscheiden fiber den notwendigen st/indigen Transfer von Stoffen und Signalen, und zwar in /iuBerst unterschiedlicher Weise, wie das etwa ffir die /iuBere und die innere Chloroplastenmembran oder die Thylakoide gut bekannt ist [1].

[CO2-fixation and translocation in benthic marine algae : I. Kinetics of (14)CO 2-assimilation in Laminaria saccharina].

When discs punched out of the median part of the phylloid of Laminaria saccharina Lamour. were exposed to H(14)CO3 (-) in the light for periods of 10 sec to 10 min, (14)C was rapidly incorporated into various photosynthetic products. As compared with dark fixation, (14)C-photosynthesis increased exponentially during the first 60 sec of incubation in H(14)CO3 (-). Fixation rates were found to be 76 μmol CO2·dm(-2)·h(-1) or 100 μmol CO2·mg(-1) chlorophyll a·h(-1). Eighty-five per cent of the total (14)C assimilated after 10 sec was fixed in phosphoglycerate and in the sugar monophosphates, 2% in the sugar diphosphates, and only 3.5% in malate and aspartate. While the radioactivity of malate and aspartate only rose to a constant level, the percentage of the total (14)C in phosphoglycerate and-to a lower extent-that in the sugar monophosphates rapidly decreased with the duration of light exposure. Simultaneously, mannitol and glycine+serine became labelled with 43% and 32% respectively of the total (14)C after 10 min light fixation. In the dark, the percentage of the total (14)C in malate decreased with the time of H(14)CO(2-)-incubation, while there was a remarkable increase in radioactivity of aspartate and glutamate. Within 60 min darkness no labelling of mannitol was found.From the present results it is concluded that the photosynthetic carbon cycle first described by Bassham and Calvin operates in Laminaria saccharina.SummaryWhen discs punched out of the median part of the phylloid of Laminaria saccharina Lamour. were exposed to H14CO3- in the light for periods of 10 sec to 10 min, 14C was rapidly incorporated into various photosynthetic products. As compared with dark fixation, 14C-photosynthesis increased exponentially during the first 60 sec of incubation in H14CO3-. Fixation rates were found to be 76 μmol CO2·dm-2·h-1 or 100 μmol CO2·mg-1 chlorophyll a·h-1. Eighty-five per cent of the total 14C assimilated after 10 sec was fixed in phosphoglycerate and in the sugar monophosphates, 2% in the sugar diphosphates, and only 3.5% in malate and aspartate. While the radioactivity of malate and aspartate only rose to a constant level, the percentage of the total 14C in phosphoglycerate and-to a lower extent-that in the sugar monophosphates rapidly decreased with the duration of light exposure. Simultaneously, mannitol and glycine+serine became labelled with 43% and 32% respectively of the total 14C after 10 min light fixation. In the dark, the percentage of the total 14C in malate decreased with the time of H14CO2--incubation, while there was a remarkable increase in radioactivity of aspartate and glutamate. Within 60 min darkness no labelling of mannitol was found.From the present results it is concluded that the photosynthetic carbon cycle first described by Bassham and Calvin operates in Laminaria saccharina.

Frond development and CO2-fixation in Laminaria hyperborea.

SummaryDiscs punched out of different zones of the found of Laminaria hyperborea (Gunn.) Fossl. were exposed to H14CO3- in the light and in the dark for various lengths of time. Photosynthetic rates in young and old parts were in the same range, 12–39 μmol CO2 dm-2 h-1 at 4°C, whereas dark fixation was remarkably higher in the growing zone than in the old frond: 13–28% of the corresponding light fixation in the young phylloid and only 2–6% in the old one. In all parts of the frond the reductive pentose phosphate cycle is the main system of carboxylation, and is accompanied by a notable primary synthesis of amino acids. In the growing zone heavy and primary labelling of malate and aspartate parallel to 3-PGA is due to the undiminshed activity of the dark fixation system (PEP-carboxykinase) in the light. Mannitol synthesis seems to be enhanced with increasing age of the phylloid, but rapid and primary 14C-incorporation into amino acids was found in all parts of the frond.The data presented here suggest a possible significance of the high dark fixation rates for the growth of the sublittoral Laminaria hyperborea. Translocation of mobilized storage material from the old phylloid might supply the young tissue with reduced material during the growth season when the light intensity is low in the habitat of this alga.

Translocation profiles of 11C-assimilates in the petiole of Marsilea quadrifolia L.

An array of three multipliers with BGO-crystals arranged along the petiole has been used to study the movement of 11C-assimilate in Marsilea quadrifolia L. The leaf blades were exposed to a 3–6 min pulse of 11CO2 under continuous light. Data were recorded with 10-s counting intervals and corrected for background, sensitivity of detectors, and 11C-decay. The speed of translocation could be obtained from the time difference between the straight lines of 11C-increase monitored at different positions along the petiole. The speed was within the range of 60 to 150 cm h-1. Cutting as well as cold treatment at definite locations on the petiole led to an immediate stop of 11C-movement. Both inhibition and—in the case of chilling—restoration of translocation were detectable within minutes. When the assimilation chamber was flushed with nitrogen gas, the normal increase of 11C at the monitored sites slowed down which, after prolonged N2-treatment, even resulted in a decrease. With a time-delay down the petiole, replacement of N2 by an air-stream led to complete restoration of the 11C-slope. These and similar shortterm effects on translocation could not be detected by former methods. This technique is also useful in with species other than Marsilea qu., which was chosen as a test plant because of the anatomical ‘simplicity’ of its petiole.

Zur Speicherung von Saccharose in der wachsenden Zuckerrübe

Summary 1. After illumination of leaves in the presence of 14 CO 2 for various times the distribution of labelled assimilates in specimens of Beta vulgaris ssp. altissima was analyzed. With increasing age the attraction of the root for sucrose exceeds that of all other growing parts of the plant. Petioles might serve as intermediate storage pools throughout the whole growth period. 2. Controlled elution of root-slices from these experiments revealed an intermediate role of the ‘free space’ in sugar transfer to the parenchyma tissue: in chase experiments the rate of eluted 14 C-assimilates decreases considerably with time. 3. In the course of the growth of the sugar-beet a distinct increase of its adenylate (uridylate-) content (per protein) was observed; energy charge raised from EC = 0.6 (at 2–5 g freshweight) to EC = 0.8 (> 800 g f. w.). At periods of unfavourable weather conditions (1974) a normal growth of the sugar-beets was apparent but the accumulation of sucrose was rather low, and energy-charge remained to be constant and decreased finally. 4. From our results it is concluded that a relationship exists between the process of accumulation and the change of energy charge. In as much as the apoplast seems to play an intermediate role in transfer of sugar from the phloem to the parenchyma cells, an hypothetical uphill-step should be localized within the storage parenchyma.

Isolierung und Eigenschaften von Mitochondrien aus Blättern von Spinacia oleracea und Beta vulgaris

SummaryFrom mature green leaves of Spinacia oleracea and Beta vulgaris a fraction has been obtained which is enriched in mitochondria. Washed leaves were crushed in a meat-mill in an isotonic or slightly hypertonic medium containing sucrose, EDTA, MgSO4, bovine serum albumin, mercaptoethanol, KH2PO4, and HEPES as a buffer substance. After squeezing through nylon the suspension was centrifuged first at 1500xg, and following removal of the sediment a second time at 25000xg. After resuspension in a washing medium, the pellet was centrifuged in a swing-out rotor for 30 min at 35000xg on a density gradient generated from a silica sol (“Ludox HS 40”). A temperature of 2°C was maintained during the whole procedure.Whereas the usual isolation techniques are unsatisfactory for the preparation of intact leaf cell mitochondria, the above procedure enabled us to obtain a fraction from the gradient which had the following properties. The respiration rate was 29.5 μatoms O/mg protein-N/h with α-ketoglutarate as a substrate. Phosphorylation rates as calculated from the phosphate measurements and P/O ratios were low. This has been interpreted as a consequence of ATPase activity in the fraction. In polarographic studies ADP/O ratios of 1.32–2.15 with succinate as a substrate were measured. Respiratory control was also observed. This indicates the presence of tightly coupled mitochondria in the fraction.The fine structure of the mitochondria was shown to be intact as far as electron microscopical evidence can be used as a criterion. The procedure appears to be suitable for isolating active mitochondria and intact chloroplasts from green leaves. It may permit the study of interrelationships between these organelles.

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10−6M fusicoccin. This effect is decreased in the presence of 10mM Na+ or K+, 2 mM Mg2+ or Ca2+. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent Km of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis−Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10−6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.