Callose Deposition and Photoassimilate Export in Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Callose accumulated on sieve plates of phloem of white bean seedlings exposed to excess Co, Ni, or Zn. The callose deposits ranged in thickness and were most pronounced in midribs of unifoliate leaves and their subtending petioles. Lesser callose deposits were found in stems. Although translocation of ¹⁴C was reduced drastically in seedlings exposed to excess metal, no correlation was found between translocated ¹⁴C and the amount of callose in the petioles. It is concluded that the inhibition of phloem translocation in seedlings exposed to excess metal is due to effects other than callose deposition.     

Temperature-induced Changes in Hill Activity of Chloroplasts Isolated from Chilling-sensitive and Chilling-resistant Plants

The effect of temperature on Hill activity has been compared in chilling-sensitive and chilling-resistant plants. The Arrhenius activation energy (Ea) for the photoreduction of 2,6-dichlorophenolindophenol by chloroplasts isolated from two chilling-sensitive plants, mung bean (Vigna radiata L. var. Mungo) and maize (Zea mays L. cv. PX 616), increased at low temperatures, below 17 C for mung bean and below 11 C for maize. However, the Ea for this reaction in pea (Pisum sativum L. cv. Massay Gem), a chilling-resistant plant, likewise increased at temperatures below 14 C. A second change in Ea occurred at higher temperatures. The Ea decreased above about 28 C for mung bean, 30 C for maize, and 25 C for pea. At temperatures approaching 40 C, thermal inactivation of Hill activity occurred. These results, when taken together with previous results obtained with the chilling-resistant plant barley, indicate that chloroplasts from both chilling-sensitive and chilling-resistant plants can undergo a change in chloroplast membrane activity at low temperatures above freezing and that the presence of such a change in chloroplast membranes is not necessarily correlated with chilling sensitivity.     

The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants.2. An Arrhenius plot of the photoreduction of NADP⁺ from water by chloroplasts isolated from tomato (Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole^?1. Between 11 and 3 °C the activation energy increases to 22 kcal·mole^?1. Photoreduction of NADP⁺ by chloroplasts from another chilling-sensitive plant, bean (Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole^?1 below about 12 °C.3. The photoreduction of NADP⁺ by chloroplasts isolated from two chilling-resistant plants, lettuce (Lactuca sativa var. winter lake) and pea (Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole^?1, respectively, over the temperature range 3–25 °C.4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP⁺ from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP⁺ reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C.5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible.6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP⁺, would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.     

OBSERVATIONS ON METAPHLOEM IN THE VEGETATIVE PARTS OF PALMS

Metaphloem was studied in available vegetative parts of 374 species in 164 genera of palms. Sieve elements usually have compound sieve plates except in the subfamilies Lepidocaryoideae and Nypoideae. Sieve elements in roots usually have oblique to very oblique end walls, whereas in stems and leaves they have transverse to oblique walls. Within a phloem strand the degree of compounding of a sieve plate is directly correlated with element diameter. Plastids are normally present in functioning, enucleate sieve elements. Small quantities of “slime” substances have been detected in young sieve elements in stems and petioles of a few species. Many sieve plates in functioning sieve elements lacked callose in materials quick-killed in liquid nitrogen or chilled acetic-alcohol. Definitive callose is confined to sieve elements just before their obliteration. Sieve tubes in leaf and stem are usually ensheathed by contiguous parenchyma cells while those in root have very few contiguous parenchyma cells. Two types of contiguous parenchyma cells can be distinguished by difference in cytoplasmic density, especially with the electron microscope. Cells with denser cytoplasm are interpreted as companion cells. Lignified contiguous parenchyma cells are occasionally present in metaphloem of petioles. The possible diagnostic and taxonomic features of metaphloem are discussed.     

Der autoradiographische Nachweis trittierter wasserlöslicher Substanzen in den Siebröhren von Cucurbita und Cucumis

Summary When D-Glucose-6-T is applied to small areas of the leaf blade of Cucumis or Cucurbita, a translocation of labelled material within the veins and the petiole down to the internodes of the axis can be demonstrated by chemical methods and by histoautoradiography. The translocated material was identified by thin layer and paper chromatography as stachyose, raffinose, and sucrose. A dry mounting autoradiographic technique was elaborated which prevented any secondary diffusion of water soluble substances. In autoradiograms of longitudinal and cross sections of veins, petioles and internodes, tritium can be localized within single sieve tubes of exporting bundles. Usually the centre of activity corresponds to the cytoplasmic material or the slime-plugs and to the sieve plates. Since sucrose, stachyose and raffinose are the main labelled translocated substances in these experiments, there is little doubt that with certain precautions such autoradiograms may present a real picture of the long-distance translocation of assimilates.

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Teil einer Dissertation unter der wissenschaftlichen Leitung von Prof. Dr.J. Willenbrink.     

Temperature dependence of action of polymyxin B on Escherichia coli

The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes. At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s. The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C. The uptake of TPP+ also increased after polymyxin addition. The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously. In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C. This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes. The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E. coli. Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability. The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.     

Distorted phytochrome action spectra in green plants

An evaluation was made of the extent which a Münch-type pressure flow mechanism (i.e., osmotically-generated pressure flow) might contribute to phloem transport in soybean. Estimates of sucrose concentrations in source (leaf) and sink (root) sieve tubes were obtained by a negativestaining procedure. Water potential measurements of the leaf and of the nutrient solution allowed calculation of the turgor pressures in source and sink sieve tubes. The turgor difference between source and sink sieve tubes was compared to that required to drive translocation at the observed velocity between the source and sink, as measured by [¹⁴C] photosynthate movement. Sieve-tube conductivity was calculated from the sieve-tube dimensions, assuming an essentially unobstructed pathway. In three experiments, the sucrose concentration was consistently higher in source sieve tubes (an average of 11.5%) than in sink sieve tubes (an average of 5.3%). The ratio of these values (2.3:1) agreed reasonably well with an earlier ratio for source/sink sieve tube concentrations of 1.8:1, obtained by quantitative microautoradiography. The resulting calculated turgor difference (an average of 4.1 bars) was adequate to drive a pressure flow mechanism at the observed translocation velocities (calculated to require a turgor difference of 1.2 to 4.6 bars). No other force need be presumed to be involved.This work was presented in part at a joint U.S.-Australian Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 15–20, 1975; see Fisher (1976)     

Transport of Kinetin-814C in Petioles

Several differences in the translocation pattern of radioactive kinetin in plant petioles were determined. Radioactivity from kinetin-8-¹⁴C (Kn*) moved from donor agar blocks through petioles of bean and cocklebur but not of cotton. There was no difference in basipetal or acropetal movement of radioactivity from Kn* in cocklebnr petioles, but there was in bean petioles. In bean petioles this movement was preferentially basipetal, but it was influenced by the age of the petiole and by the presence of added indoleactic acid. The combination treatment accelerated the basipetal movement of radioactivity from Kn* in young bean petioles and not in old ones. All data is based on radioactivity translocated into receiver agar blocks which were assayed individually in a liquid scintillation spectrometer. The results show that plant species, direction of transport, age of tissue, and presence of IAA can all influence the translocation of Kn* in petioles.     

Action potentials in maize sieve tubes change phloem translocation

The inhibition of phloem translocation caused by electric- andcold-shock was studied by macro- and microautoradiography inmature leaves of maize (Zea mays L.). In addition, both typesof stimulation triggered action potentials with amplitudes ofmore than 50 mV which were transmitted without diminution insieve tubes with velocities of 3-5 cm s^-1. By utilizing X-raymicroanalysis ion concentrations of sieve tubes were measuredin non-stimulated and stimulated leaves. It was shown that potassiumand chloride diminished about 3-fold after stimulation whilethe amount of cytoplasmic calcium may have increased. Thesedisplacements lead to the conclusion that calcium influx aswell as potassium and chloride efflux are involved in the propagationof action potentials. A possible relationship between electricsignalling and the reduction of phloem translocation is discussed. Key words: Action potential, electric and cooling stimuli, energy-dispersive X-ray microanalysis, ion shifts, phloem translocation     

The Fine Structure of the Sieve Tubes of the Petiole of Nytnphoides peltatum (Gmel.) O. Kunze

A study has been made by electron microscopy of the fine structureof the peti-olar sieve tubes of the water plant Nymphoides peltatum.These are found to have very well-developed nacreous walls.The pores of the sieve plates appear to be filled in functioningsieve tubes with densely staining cytoplasm. The peripheralcytoplasm of the sieve tubes seems to contain an extensive developmentof the endoplasmic reticulum, whose elements become finer nearthe plates and crowd together through the pores. These findingsappear to be compatible with more than one theory of translocation,including the electro-osmotic theory of mass flow.     

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade‐off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier–Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.     

Effect of Water Stress on Turgor Differences and C-Assimilate Movement in Phloem of Ecballium elaterium

The effects of water stress on pressure differences and ¹⁴C-assimilate translocation in sieve tubes of squirting cucumber Ecballium elaterium A. Rich were studied. Water stress was induced by transfer of plants from culture solution to a polyethylene glycol 6,000 solution having an osmotic potential of −18.2 atm. Sieve tube turgor, turgor differences between source and sink, and translocation rate were decreased. After 260 minutes of translocation, only 19% of the total fixed ¹⁴CO₂ had moved out of the leaf, compared to the control value of 62% after the same period of time. The results suggest that water stress slows translocation by lowering sieve tube turgor differences, which are essential for the pressure flow mechanism of conduction.     

Search for an endotherm in chloroplast lamellar membranes associated with chilling-inhibition of photosynthesis

The phase transition of chloroplast lamellar membrane lipids has been proposed to be the underlying cause of chilling-induced inhibition of photosynthesis in sensitive plants. Differential scanning calorimetry has been used to search for any endotherms arising from lipid state changes in chloroplast lamellar membranes of the chilling-sensitive plants cantaloupe , kidney bean, domestic tomato, and soybean. For comparison, calorimetric scans of chloroplast lamellar membranes from the chilling-insensitive plants spinach, pea, and wild tomato were made. A large reversible endotherm, extending from below 10 degrees to nearly 40 degrees C, was observed in chloroplast membranes from tomatoes of both chilling-sensitive (Lycopersicon esculentum Mill. cv. Floramerica ) and chilling-insensitive (L. hirsutum LA 1361) species. A much smaller endotherm, approximately 5 to 10% of the area of that seen in the two tomato species, and extending over a similar temperature range, was detected in chloroplasts from chilling-insensitive spinach and peas, and also was generally observed in chloroplasts from chilling-sensitive cantaloupe , kidney bean, and soybean. The enthalpy of these smaller endotherms indicates that, if the endotherm arose entirely from a lipid transition, then it corresponded to the melting of less than about 10% of the total membrane polar lipid. On the basis of these data it is concluded that there is no correlation between chilling sensitivity of photosynthesis and the presence or absence of a phase transition of bulk membrane lipids of the chloroplast lamellar membrane at temperatures above 5 degrees C.     

Some Low-temperature Effects on Sieve Tube Translocation in Salix viminalis

The data presented on translocation of ¹⁴C-labelled compoundsin Salix viminalis show that above a temperature close to –4°C translocation occurs, whilst below –4 °C inmost cases it does not. Stoppages were irreversible when the temperature of the cooledstem was raised again to normal (approximately 20 °C) andappeared to involve some disruption of the phloem. The temperaturesat which the bark was found to freeze were found to be closelysimilar, with respect to level and variability, to the stoppingtemperature. Respiration measurements on isolated strips of bark at –2°C showed that oxygen uptake fell to approximately 5 percent of its value at 25 °C. The decreased level of radioactivity in the cooled regions ofthe stems is considered to be evidence that the exchange oflabelled compounds between the sieve tubes and the surroundingcells was slowed up at low temperatures.     

Ricerche Sull'ultrastruttura dei Tubi Cribrosi di Piccioli di Foglie di Edera

Abstract

The ultrastructure of sieve tubes in leaf petioles of HEDERA HELIX. — The structural organization of the sieve elements in Hedera leaf petiole at the beginning of the second year of life has been studied. At this stage of life the sieve tubes are completely developed, but still in full activity.

Their plasmatic structures, though altered, show that they are still alive. The cytoplasm forms a parietal layer; mitochondria, endoplasmic reticulum and plastids are present although very peculiar in aspect. The cytoplasm is bounded externally by a plasmalemma; on the contrary no tonoplast is detectable.

The data reported in this paper are favourable to the idea of an active partecipation of the sieve tubes in the translocation of organic solutes, in agreement with the findings concerning the oat coleoptile.     

Sequence of morphological and physiological events during natural ageing and senescence of a castor bean leaf: sieve tube occlusion and carbohydrate back-up precede chlorophyll degradation

The development of castor bean ( Ricinus communis L. var. sanguineus) leaves from bud break to abscission was studied to determine whether senescence of phloem precedes or follows chlorophyll degradation in the course of natural ageing of leaves. The castor bean leaf blade took 20 days for full expansion and its average life span was 60 days. From the day of full expansion on it suffered a substantial loss in N, a small loss in C, K and P and a gain in Ca, Mg and S. The content of soluble sugars increased with time, paralleled by a decrease of photosynthetic activity. Starch accumulated shortly before chlorophyll breakdown. The amino acid level in the leaves decreased steadily together with nitrate reductase and glutamine synthetase activity. Reactive oxygen species increased and oxidation-protecting compounds decreased during the life span of the leaves. Shortly after full leaf expansion an increasing number of sieve plates showed strong callose depositions when visualized by aniline blue method. At day 40 only half of the sieve tubes appeared functional. Chlorophyll breakdown followed these processes with a time lag of approximately 10 days. The sieve tube sap of ageing leaves had the same sucrose concentrations as young leaves, whereas amino acid concentrations decreased. High levels of reduced ascorbic acid and glutathione together with increasing levels of glutaredoxin indicated oxidative strain during senescence. We speculate that the gradual increase of reactive oxygen species during ageing together with the import of calcium ions lead to the stimulation of callose synthesis in plasmodesmata and sieve plates with the consequence of inhibition of phloem transport leading to carbohydrate back-up in the leaf blade. The latter may finally induce chlorophyll breakdown and, at the end, leaf abscission at the petiole base. Thus phloem blockage would precede and may be causal for chlorophyll degradation in leaf senescence.     

Mechanism of cyanide inhibition of Phloem translocation

Petiolar application of potassium cyanide inhibited ¹⁴C-assimilate translocation without affecting source leaf photosynthesis or phloem loading of sucrose in Phaseolus vulgaris. The inhibition of transport was correlated with disruption of the structural integrity of the sieve tubes (sieve pore blockage) rather than impairment of a metabolic process in the translocation path driving translocation.     

Carbohydrate translocation in sugar beet petioles in relation to petiolar respiration and adenosine 5'-triphosphate

Earlier studies have shown that the retarding effect of low petiolar temperatures on sucrose transport through sugar beet (Beta vulgaris L.) petioles is markedly time-dependent. Although the initial effect of chilling the petiole to near 0 C is severely inhibitory, translocation rates soon recover (usually within about 2 hours) to values at or near the control rate. In the present studies, selected metabolic parameters were measured simultaneously with translocation. No stoichiometric relationships among petiolar sucrose transport, petiolar respiration (CO₂ production), and calculated petiolar ATP turnover rates were evident. It appears that the major sources of energy input energizing carbohydrate transport in sieve tubes function mainly at either loading or unloading sites and not at the level of individual sieve-tube elements.     

An investigation of bidirectional translocation in the Phloem

Patterns of (14) CO(2) , assimilate movement in Vicia jaba plants having 7 nodes were studied. Bidirectional translocation occurred throughout most of the stem length when tracer was applied to leaves of various ages. To determine whether this bidirectional translocation occurs within single sieve tubes, a O.1 % solution of the fluorescent dye K-fluorescein was applied to a lightly scraped area on the stem in the middle of a young internode. After one hour the dye was present short distances above and below the treated area. Free-hand sections of the internode showed the dye to be localized in the traces of the larger leaves below tbe treated area and in the traces of the younger leaves above the treated area. The dye was never present in the same bundle both above and below the treated area, indicating that each bundle and sieve tube translocated the dye in only one direction. These results were confirmed using Phaseolus vulgaris, Vinca rosea, and Pelargonium hortum. A similar study in which petioles of young Ecballium elaterium leaves were treated showed that usually the phloem of one bundle translocated the dye in only one direction but in some cases the external phloem of the bicollateral bundles carried the dye toward the stem while the internal phloem carried the dye toward the blade. When longer time intervals were used in all these experiments, the dye sometimes appeared in the same phloem areas both above and below the treated area. This is explained by a lateral transfer of tracer within the phloem, either through secondary phloem or through bundle anastomoses at the nodes.     

Temperature dependence of delayed ehlorophyll fluorescence in intact leaves of higher plants. A rapid method for detecting the phase transition of thylakoid membrane lipids

The temperature dependence of the yield of in vivo prompt and delayed chiorophyll fluorescence was investigated in maize and barley leaves. In the chilling-sensitive maize, delayed fluorescence at steady-state level showed a maximum near the temperature at which thylakoid membrane lipids undergo a phase transition as revealed by differential scanning calorimetry measurements. In the chilling-resistant barley, no phase transition was detected above 0°C and the delayed light emission varied in a monotonic fashion. It was shown that measurements of delayed luminescence intensity in vivo can provide a rapid and sensitive method for detecting the phase change of membrane lipids in intact leaves of chilling-sensitive plant species such as tomato, cotton, cucumber, castor bean or avocado. In contrast, the use of steady-state prompt chlorophyll fluorescence as an indicator of membrane fluidity change was not successful.