Vein Loading in Seedlings of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Vein loading in unifoliate leaves of white bean seedlings exposed to excess Co²⁺, Ni²⁺, or Zn²⁺ for 1 to 4 days was studied by incubating leaf discs in [¹⁴C]sucrose. The discs from plants exposed to metal exhibited an increased total uptake of radiosucrose but reduced vein loading. Differences between treatments due to infiltration of disc margins were eliminated by analyzing 7-millimeter discs cut from the center of incubated 14-millimeter discs. Uptake of radiosucrose was greater particularly in discs from seedlings exposed to excess Ni²⁺ and Zn²⁺. The effect increased as exposure of the seedlings to metal increased up to 4 days. Autoradiographs showed vein loading in control leaf tissues with most of the radiosucrose accumulating in minor veins and little remaining in the mesophyll. In discs from metal-treated plants, most of the sucrose remained in the mesophyll without accumulating preferentially in the minor veins. This effect was evident within 24 hours of exposure to excess metal and intensified with longer exposures to metal. The inhibition of vein loading was also evident in situ. Both the preferential accumulation of sucrose into the minor veins of control tissues and the accumulation into mesophyll of metal exposed tissues were sensitive to 2,4-dinitrophenol and the blockage of sulfhydryl groups. It is concluded that the inhibition of vein loading contributes markedly to the observed toxicological effects of reduced photoassimilate export and of accumulation of carbohydrates in fully expanded leaves of bean seedlings exposed to excess metal ions.     

The Effect of 2,4-Dinitrophenol on Translocation in the Phloem

The effect of 2,4-dinitrophenol (DNP) on sucrose-¹⁴C transport in Soya seedlings has been analysed. The aim was to distinguish between an effect of the inhibitor on sugar movement within the phloem sieve tubes themselves, and on the prior steps of uptake and secretion of sugar into the conducting cells. DNP drastically inhibited sucrose-¹⁴C transport if it was applied to the ¹⁴C-treated leaf immediately before, or during, ¹⁴C supply. Transport was also strongly inhibited if DNP was applied along the translocation path while the ¹⁴C-treated leaflet was still in position on the plant. When, bowever, DNP was applied through the cut petioles of the primary leaves after removal of the ¹⁴C-treated terminal leaflet of the first trifoliate leaf, no inbibition was observed. On the contrary, transport appeared to have been promoted: significantly more ¹⁴C disappeared from the upper regions of DNP-treated plants as compared with controls, while in the lower plant parts more ¹⁴C accumulated. Different rates of synthesis of sucrose-¹⁴C into non-alcohol-soluble compounds could not account for this result. A similar stimulatory effect was observed when DNP was applied to the cut petiole of a primary leaf opposite that treated with ¹⁴C. Several indications were obtained that ¹⁴C which has reached the lower parts of the plant may circulate upwards again through the phloem within about 15 minutes. When sucrose-¹⁴C was introduced into the roots via the xylem, both DNP treatment and prior steam girdling resulted in the apparent accumulation of ¹⁴C in the lower plant parts. the results would be compatible with DNP inhibition of upwards movement in the phloem. DNP might also have affected sugar uptake processes in cells neighbouring the translocation path. It is concluded that the inhibitory effect of DNP on downwards phloem transport reported by earlier workers was probably due to an effect on uptake and/or secretion into the sieve tubes, not to an effect on the conducting cells themselves. Modern theories for phloem transport are discussed in the light of these findings.     

Enhancement of Phloem Exudation from Fraxinus uhdei Wenz. (Evergreen Ash) using Ethylenediaminetetraacetic Acid

Ethylenediaminetetraacetic acid (EDTA) enhanced the exudation of ¹⁴C-labeled assimilates from excised leaflets and whole plant specimens of Fraxinus uhdei Wenz. A 2 millimolar EDTA concentration was found to be most effective in promoting exudation from excised leaflets, while 10 millimolar EDTA was most effective in whole plants experiments. Exudation rate reached a maximum after 24 hours in both experiments. The continuous presence of EDTA throughout the treatment period was required for maximum exudation from excised leaflets. Stachyose, raffinose, verbascose, and sucrose were the principal compounds found to occur in exudate samples. These compounds are typically transported in sieve elements of various Fraxinus species suggesting the exudate was of phloem origin. Electron microscope studies of petiolule sieve plate pores from excised leaflets showed substantially less callose appearing after treatment with EDTA than after H₂O treatment. It is suggested that EDTA enhances phloem exudation by inhibiting or reducing callose formation in sieve plate pores. The exudation enhancement technique described for whole plant specimens is suggested as a useful means of collecting phloem sap and studying translocation in woody plants.     

The contribution of tonoplast and plasma membrane to the electrical properties of a higher-plant cell

Translocation of ¹⁴C-labelled assimilates down the petioles was studied in intact plants of Pelargonium zonale (L.) L'Hérit ex Ait. The central bundle of the petiole was dissected out and treated with solutions of various inhibitors. Whereas cytochalasin B had no effect on ¹⁴C-translocation, a distinct and localized inhibition was caused by CCCP (10^-7 M), antimycin (5×10^-5 M), atractylate (5×10^-5 M), and valinomycin (10^-5 M) without any significant change in the proportion of [¹⁴C]sucrose in the translocate. The inhibition of translocation is inferred both by accumulation of ¹⁴C distal to and a decrease in ¹⁴C concentration basal to the treated petiolar region. If valinomycin was fed into the transpiration stream by flapping the peripheral bundles of the petioles an increased labeling of sugar phosphates occurred in the ¹⁴C fed leaf. Plasmolysis tests indicated that whereas CCCP interfered with the semipermeability of phloem cell membranes, valinomycin had no such effect. The results with valinomycin suggest a compartmentation of potassium ions for the translocation process but are ambiguous as to whether or not a potassium pump is involved.Dedicated to Wilhelm Halbsguth, Kiel to his 65th birthday     

Adenosine triphosphatase in the phloem of Cucurbita

Summary The distribution of adenosine triphosphatase (ATPase) activity in the phloem of petioles and minor veins of Cucurbita maxima has been studied using a lead phosphate precipitation procedure. ATPase activity was localized in sieve elements, companion cells and parenchyma cells. Activity was found at the cell surfaces, associated with the dispersed P-protein of mature sieve elements, in mitochondria, sieve-element reticulum, and at specific regions of the cell walls. It is suggested that the ATPase at the phloem cell surfaces may function in intercellular transport of assimilates or ions, and that the ATPase activity associated with the P-protein may function in the translocation process or in callose deposition.     

Uptake and Utilization of Xylem-borne Amino Compounds by Shoot Organs of a Legume

Amino compounds representative of the major N solutes of xylem sap were pulse-fed (10 to 20 minutes) singly in ¹⁴C-labeled form to cut transpiring shoots of white lupin (Lupinus albus L.). ¹⁴C distribution was studied by autoradiography and radioassays of phloem sap, leaflet tissues, and shoot parts harvested at intervals after labeling. Primary distribution of N by xylem was simulated using a 20-minute labeling pulse followed by a 30-minute chase in unlabeled xylem sap. Shoots fed ¹⁴C-labeled asparagine, glutamine, valine, serine, or arginine showed intense labeling of leaflet veins and marked retention (35 to 78%) of ¹⁴C by stem + petioles. Shoots fed ¹⁴C-labeled aspartic acid or glutamic acid showed heaviest ¹⁴C accumulation in interveinal regions of leaflets and low uptake (11 to 20%) of ¹⁴C by stem + petioles. Departing leaf traces were major sites of uptake of all amino compounds, and the implications of this were evaluated. Fruits acquired only 1 to 5% of the fed label directly from xylem, but more than doubled their intake during the period 30 to 160 minutes after feeding through receipt of ¹⁴C transferred from xylem to phloem in stem and leaves. ¹⁴C-Labeled asparagine and valine transferred directly from xylem to phloem, but the ¹⁴C of ¹⁴C-labeled aspartic acid and arginine appeared in phloem mainly as metabolic products of the fed compound. The labeling of the soluble pool of leaflets reflected these differences. The significance of heterogeneity in distribution and metabolism of xylem amino compounds in the shoot was discussed.     

6(5)CARBOXYFLUORESCEIN AS A TRACER OF PHLOEM SAP TRANSLOCATION

6(5)carboxyfluorescein (6(5)CF), a polar fluorescein with an apparent pK of 6.3, was introduced, as a pH 6.3 solution, into the apoplast of lamina or petioles of mature soybean leaves. Freehand sections were prepared at various times and immediately observed with a fluorescence microscope. 6(5)CF-associated fluorescence appeared in all sink organs, from shoot apex to roots. It was strictly confined to the phloem regions, even after 4 days. Its transport into young leaves ceased at approximately the time they underwent sink-to-source transition. It was never transported between two leaflets of the same leaf. Its transport was interrupted by phloem destruction. All these transport characteristics were highly reproducible, and were paralleled by those of ¹⁴C transport after application of (¹⁴C)sucrose to leaf surfaces. In contrast with 6(5)CF, fluorescein was transported between mature leaves, and between leaflets of the same leaf. It was not restricted to phloem, and often appeared in the xylem region. These results indicate that 6(5)CF can be used to monitor phloem sap translocation in real time, in short- and long-term experiments.     

Untersuchungen zur funktionellen Anatomie des Leitgewebesystems im Blatt von Pelargonium zonale

Summary The petiole of Pelargonium zonale is traversed by 17 bundles, whose arrangement and form are typical for this plant. The bundles of the petiole are connected with the conducting system of the axis and with the main nerves by a system of phloem anastomoses in the leaf base and in the junction between the petiole and the leaf blade (Fig. 2). The anatomical findings were confirmed and extended by a study of the translocation of K-fluorescein and ¹⁴C. It could be shown that the metaphloem of the central petiole bundle is composed of phloem subunits, each of which is connected with the phloem of one certain main nerve only (Fig. 4). Accordingly, if fluorescein or ¹⁴CO₂ is applied to one main nerve, the dye or ¹⁴C-material is translocated exclusively in a small phloem area of the central bundle. Autoradiograms of the petioles indicate that the ¹⁴C-labelled assimilates (sucrose, glucose, fructose and amino acids) are translocated exclusively in the phloem. A lateral movement of the labelled material within the petiole was not observed. The metaphloem of the central petiole bundle of Pelargonium zonale revealed a functional organization of phloem subunits.

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

Phloem transport in Picea abies (L.) Karst. in mid-winter

Summary Translocation of ¹⁴C assimilates was studied on four different transport systems of Picea abies branches after induced activation in January. ¹⁴CO₂ assimilation of terminal shoots for 48 h at 25° C resulted in phloem loading and basipetal transport of ¹⁴C photosynthate into the following, older shoot generations. ¹⁴C import was enhanced, when these older shoot generations were kept in the dark. Microautoradiographs of the labelled terminal shoots showed that ¹⁴C assimilates were exported from needles via sieve elements of the leaf traces and loaded into the latest increment of the axial secondary phloem. No ¹⁴C label appeared in the obliterated sieve cells or in the tracheids. In addition, ¹⁴C photosynthate accumulated densely in the chlorophyllous cells of the cortex and in cells of the resin ducts, indicating certain sink activity. In the darkened 2-year-old shoot, imported ¹⁴C photosynthate was concentrated in the functional secondary phloem, while some ¹⁴C label was unloaded into the latest xylem increment. When 6-year-old shoots were exposed to ¹⁴CO₂ for 48 h in the light, ¹⁴C assimilates accumulated in the phloem of the leaf trace and in the latest increment of the axial secondary phloem. However, a substantial amount of radioactivity was unloaded into ray cells and phloem parenchyma cells. Thus, the presence of functioning phloem in needles and twigs of P. abies during winter allows long-distance translocation and radial distribution of assimilates according to existing source-sink relations.     

Photoassimilate translocation in the petiole of Cyclamen and Primula is independent of lateral retrieval

Phloem translocation of photoassimilates between source andsink is considered to be linked with active retrieval of sugarsleaked to the vascular apoplast. This hypothesis was evaluatedby studying photo-assimilate movement in petioles of intactplants of Cyclamen persicum and Primula obconica in the presenceof inhibitors affecting sucrose retrieval (PCMBS, CCCP). Inhibitorsolutions were applied by rinsing locally isolated petiole bundlesor by injection into the petioe parenchyma. PCMBS and CCCP reduced[¹⁴C]sucrose retrieval from the petiole apoplast by the vascularcells and altered the distribution pattern of ¹⁴C-photoassimilateswithin the petiole tissues. However, these treatments did notaffect translocation through the petiole phloem. Evidence isprovided that the reagents were present in the vascular apoplastsurrounding the translocating phloem. It was concluded thatassimilate movement in the petiole of Cyclamen and Primula wasindependent of apoplastic retrieval. Key words: Cyclamen, Primula, phloem, transport, path, sucrose, retrieval     

Characterization of phloem exudation from castor-bean cotyledons

Exudate was collected fromRicinus communis L. cotyledons after cutting the hypocotyl. It contained high levels of sucrose and potassium, a low level of calcium, and a pH of approx. 7.5. After application of [¹⁴C] sucrose to the cotyledons, radioactivity could be recovered from the exudate, indicating that the exudate was derived from the phloem. Using data from a number of individual seedlings, correlations between loading rates of sucrose, translocation rates, and sucrose and potassium contents were analyzed. A positive correlation was found between the rate of sucrose loading and the rate of sucrose exudation, whereas a negative correlation existed between the contents of sucrose and potassium in the phloem.     

Carbohydrate Levels and Photoassimilate Export from Leaves of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Exposure of white bean seedlings to phytotoxic burdens of Co, Ni, or Zn reduced the export of ¹⁴C-photoassimilates from the nearly fully expanded unifoliate leaves. Little ¹⁴C reached the major sink areas, the young trifoliate leaves and the root tips, of seedlings exposed to metal. The unifoliate leaves accumulated sucrose, reducing sugars, and starch. These effects were evident within 1 or 2 days.     

Comparison of ethylenediaminetetraacetate-enhanced exudation from detached and translocation from attached bean leaves

A technique for collection of phloem exudate from detached leaves using 20 millimolar EDTA (pH 7.0) has previously been developed (King, Zeevaart 1974 Plant Physiol 53: 96-103). It was the aim of the present study to determine the efficiency of this technique in relation to undisturbed export from attached leaves. Paired primary leaves of bean seedlings (Phaseolus vulgaris L. cv Montcalm) were used to minimize variations in plant material. Attached leaves, exposed to ¹⁴CO₂ for 10 minutes with subsequent excision of one of the leaves and collection of the exudate over a 12-hour period, showed a 25% export of total assimilated ¹⁴C from the attached versus 15% of total assimilated ¹⁴C in the form of exudation from the detached ones. Leaf excision changed the labeling pattern within the leaf, increasing% total leaf ¹⁴C-activity in the ethanolic fraction, while decreasing activity in the starch fraction, as compared to attached leaves. This was presumably caused by a lack of translocation from the detached leaves. Excision did not affect dark respiration. However, measurements of total nonstructural carbohydrates in leaf starch and neutral fractions indicated no significant differences between attached and leaves detached in EDTA. Thus, in terms of actual carbon export, and accompanying distribution of nonexported carbohydrate within the leaf, EDTA-enhanced exudation compares favorably with translocation from attached leaves.     

Effect of nitrate infusion into the shoot apoplast on photosynthesis and assimilate transport in symplastic and apoplastic plants

The shoots of fireweed (Chamerion angustifolium (L.) Holub) and common flax (Linum usitatissimum L.) were infused with 50 mM KNO₃ solution to compare the influence of nitrate on photosynthesis and assimilate export from leaves in plants with the symplastic and apoplastic phloem loading, respectively. The infusion of nitrate in the shoots of both plant species lowered ¹⁴CO₂ fixation and enhanced the assimilate transport in the upward direction. Irrespective of the phloem loading type, the incorporation of ¹⁴C into sucrose decreased in nitrate-treated seedlings exposed to assimilation for short (3 min) periods. However, when shoots were sampled 3 h after ¹⁴CO₂ fixation, the content of ¹⁴C-labeled sucrose was higher in treated plants than in control seedlings infused with water. In fireweed, in contrast to flax, a similar temporal pattern was also characteristic for ¹⁴C incorporation into oligosaccharides. Within 3 h after nitrate infusion into the fireweed apoplast, the mitochondria and the cell vacuolar system underwent ultrastructural changes indicative of the increase in cytosolic osmotic pressure. At the same time, we observed accumulation of fibrillar inclusions in cell vacuoles of vascular bundles. It is concluded that the mechanisms of nitrate influence on photosynthesis and sugar export in leaves of symplastic and apoplastic plants are similar to a certain extent and involve the blocking of pores in phloem tubes, initiated by the NO-signaling system.     

THE EFFECT OF BORON DEFICIENCY ON CALLOSE FORMATION AND 14C TRANSLOCATION IN BEAN (PHASEOLUS VULGARIS L.) AND COTTON (GOSSYPIUM HIRSUTUM L.)

Callose accumulated in the tissues of boron deficient bean and cotton plants, the extent and distribution of which depended on the species. Sieve plates in the phloem of boron deficient bean were characterized by heavy plugs of callose, while the sieve plates of boron deficient cotton were essentially unaffected. Translocation of ¹⁴C was, however, drastically reduced in both plants. It is suggested that callose deposition in boron deficient plants is a secondary effect of cellular damage.     

Translocation and accumulation of translocate in the sugar beet petiole

Accumulation of translocate during steady-state labeling of photosynthate was measured in the source leaf petioles of sugar beet (Beta vulgaris L. monogerm hybrid). During an 8-hr period, 2.7% of the translocate or 0.38 μg carbon/min was accumulated per cm petiole. Material was stored mainly as sucrose and as compounds insoluble in 80% ethanol. The minimum peak velocity of translocation approached an average of 54 cm/hr as the specific activity of the ¹⁴CO₂ pulse was progressively increased. The ratio of cross sectional area required for translocation to actual sieve tube area in the petiole was 1.2. A regression analysis of translocation rate versus sieve tube cross sectional area yielded a coefficient of 0.76. The specific mass transfer rate in the petiole was 1.4 g/hr cm² phloem or 4.8 g/hr cm² sieve tube. Histoautoradiographic studies indicated that translocation occurs through the area of phloem occupied by sieve tubes and companion cells while storage occurs in these cells plus cambium and phloem parenchyma cells. The ability of the petiole to act as a sink for translocate is consistent with the concept that storage along path tissue serves to buffer sucrose concentration in the translocate during periods of fluctuating assimilation.     

Phosphoenolpyruvate carboxykinase in cucumber plants is increased both by ammonium and by acidification,and is present in the phloem

In cucumber (Cucumis sativus L.), phosphoenolpyruvate carboxykinase (PEPCK) was shown by activity measurements and immunoblots to be present in leaves, stems, roots, flowers, fruit and seed. However, immunolocalisation showed that it was present only in certain cell types. PEPCK was present in the companion cells of the adaxial phloem of minor veins, the adaxial and abaxial phloem of larger veins, the internal and external phloem of vascular bundles in petioles and stems, the phloem in roots and the extra-fascicular phloem in leaves, cotyledons, petioles and stems. Immunohistochemical evidence suggests that both the extra-fascicular phloem and the adaxial phloem are involved in the transport of amino acids. In roots and stems, the abundance of PEPCK was greatly increased by watering plants with a solution of ammonium chloride at low, but not at high pH. PEPCK also increased in leaves, but not roots or stems, of seedlings grown in an atmosphere containing 5% CO₂, and in roots and stems of seedlings watered with butyric acid. All these treatments are known to lower the pH of plant cells. Amino acid metabolism in the phloem may produce an excess of carbon skeletons, pH perturbations and an imbalance in the production/utilisation of NADH. This raises the possibility that PEPCK may function in the conversion of these carbon skeletons to PEP, which, depending on the energy requirements of the phloem, is subsequently utilised by either gluconeogenesis or the Krebs cycle, which both consume protons.Abbreviations Asp Aspartate - Asn Asparagine - Glu Glutamate - Gln Glutamine - NADP-ME NADP-malic enzyme - OAA Oxaloacetate - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - PEPCK Phosphoenolpyruvate carboxykinase     

Influence of UV-B Radiation on Early Seedling Growth and Translocation of 65Zn from Cotyledons in Cotton

The influence of UV-B radiation from filtered or unfiltered fluorescent sunlamps on early seedling growth and translocation of ⁶⁵Zn from cotyledons to the shoot was examined in two cultivars of cotton, Acala and Gregg. Ten-day-old seedlings which had been irradiated in the greenhouse for 6 h continuously each day for 14 days with 0.81 or 1.61 W × m^-2 UV-B radiation under two unfiltered FS-40 sunlamps, showed pronounced phytotoxic damage. This was characterized at first by bronzing and glazing of the cotyledons and later by upward curling of the leaves and abscission. Leaf expansion, dry matter accumulation, and mobilization of ⁶⁵Zn from the cotyledons was severely impaired in the young developing shoot under unfiltered UV-B radiation. A significant stress response also was observed in seedlings exposed to 0.61 W × m^-2 UV-B radiation through a polystyrene filter and 0.73 W × m^-2 UV-B radiation through a cellulose-acetate filter. This stress response was characterized by the formation of a red pigment in the petioles of the cotyledons, reduced leaf expansion, and reduced transport of ⁶⁵Zn. Control seedlings exposed to 0.03 W × m^-2 UV-B radiation through a mylar filter were green, had maximum leaf size and dry-matter accumulation, and had the greatest percentage of ⁶⁵Zn translocated from the cotyledons.     

Transport of sucrose, not hexose, in the phloem

Several lines of evidence indicate that glucose and fructose are essentially absent in mobile phloem sap. However, this paradigm has been called into question, especially but not entirely, with respect to species in the Ranunculaceae and Papaveraceae. In the experiments in question, phloem sap was obtained by detaching leaves and placing the cut ends of the petioles in an EDTA solution. More hexose than sucrose was detected. In the present study, these results were confirmed for four species. However, almost identical results were obtained when the leaf blades were removed and only petiole stubs were immersed. This suggests that the sugars in the EDTA solution represent compounds extracted from the petioles, rather than sugars in transit in the phloem. In further experiments, the leaf blades were exposed to (14)CO(2) and, following a chase period, radiolabelled sugars in the petioles and EDTA exudate were identified. Almost all the radiolabel was in the form of [(14)C]sucrose, with little radiolabelled hexose. The data support the long-held contention that sucrose is a ubiquitous transport sugar, but hexoses are essentially absent in the phloem stream.     

The effect of irradiance, p-chloromercuribenzenesulphonic acid and fusicoccin on the long distance transport in Zea mays L. seedlings

The system consisting of a few proportional detectors with appropriate electronic components was earlier developed for in vivo studies of long distance transport in whole maize seedlings. ¹⁴CO₂ assimilation rate (P_a), time of radioactivity appearing in the loading region (AT), transport speed in the leaf (TS_l), transport speed between the leaf and the roots (TS_r), the maximum radioactivity values detected in the leaf below the feeding area (R_l) and in the mesocotyl (R_r) from leaves to roots in maize seedlings were calculated from the obtained temporal profiles of radioactivity. The study was undertaken to follow the changes in separate steps of long distance transport in maize seedlings as affected by two light irradiances and application of p-chloromercuribenzenesulphonic acid and fusicoccin, with the aim to investigate different steps of long distance transport, particularly phloem loading. The method used allows to study in vivo the different aspects of long distance transport in maize seedlings, both qualitatively and quantitatively. It was shown that the characteristics obtained from the radioactivity profiles corresponded to different steps of long distance transport, as assimilate synthesis, phloem loading, and phloem translocation. It was also demonstrated that although active phloem loading participate in assimilate export from the leaves, assimilate transport along the maize seedling might undergo accordingly to assimilate gradient, particularly under light irradiance higher than during the growth.