Characteristics of the phloem path: analysis and distribution of carbohydrates in the petiole of Cyclamen

Compartmentation fluxes of carbohydrates along the phloem path were analysed in the petiole of Cyclamen persicum (L.) Mill. Sucrose represented the dominant fraction (58-75% of soluble carbohydrates in the vascular symplast). Planteose (12-22%), glucose (3-8%) and fructose (3-13%) occurred in lower amounts (data from liquid chromatography, percentages of the total peak area). Starch was not detectable. Upon feeding leaves with ¹⁴CO2, 98% and 90% of radiolabel was recovered as sucrose in the vascular symplast after 3 h and 24 h, respectively. Thus, sucrose appeared to be the exclusive transport sugar in Cyclamen. Experiments with asymmetrically labelled sucrose revealed that there was no metabolism of translocated sucrose. Analysis of six consecutive petiole segments (each 2 cm in length) showed a homogeneous longitudinal distribution of these sugars differed markedly. On average, the sucrose concentration amounted to 4.7 and 0.4 mg g^-1 FM in the vascular apoplast and petiole parenchyma, respectively. Sucrose was unloaded with out hydrolysis and stored in the periphery of the phloem path. Planteose was identified as another storage saccharide. Sucrose synthesis by sucrose phosphate synthase occurred when isolated vascular bundles were incubated with [¹⁴C]glucose or [¹⁴C]fructose. These data suggest that the phloem path is characterized by both source and sink like activity.     

RADIOAUTOGRAPHIC STUDY OF TRANSLOCATION IN BEAN LEAVES

The movement of the radioactivity from sucrose, indole-3-acetic acid (IAA) and phosphate has been examined in excised bean leaves. Preferential translocation of the labelled materials toward the base of leaf and petiole was demonstrated, suggesting a natural mobilization gradient down the leaf and petiole. Establishment of other mobilization centers in the leaf by local application of N⁶-benzyladenine diverted the movement of the sucrose label and, to a lesser extent, the phosphate label. There was no apparent mobilization of IAA by benzyladenine. Evidence is provided that there is a continuity of label from the source to the sink regions, and it is suggested that reported instances of noncontinuity of label may be attributable to the refixation of respired C¹⁴O₂ by tissue treated with benzyladenine. The observations appear to substantiate the concept that the unloading of solutes from the phloem can regulate the direction and intensity of translocation.     

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     

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.     

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 loading in peach: Symplastic or apoplastic?

Sorbitol and sucrose are the two main soluble carbohydrates in mature peach leaves. Both are translocated in the phloem, in peach as in other rosaceous trees. The respective role of these two soluble carbohydrates in the leaf carbon budget, and their phloem loading pathway, remain poorly documented. Though many studies have been carried out on the compartmentation and export of sucrose in sucrose-transporting species, far less is known about sorbitol in species transporting both sucrose and sorbitol. Sorbitol and sucrose concentrations were measured in several tissues and in sap, in 2-month-old peach (Prunus persica L. Batsch) seedlings, i.e. leaf blade, leaf main vein, petiole, xylem sap collected using a pressure bomb, and phloem sap collected by aphid stylets. The sorbitol to sucrose molar ratio depended on the tissue or sap, the highest value (about 7) found in the leaf main vein. Sorbitol concentration in the phloem sap was about 560 mM, whereas that of sucrose was about 140 mM. The lowest sorbitol and sucrose concentrations were observed in xylem sap collected from the shoot. The volume of the leaf apoplast, estimated by infiltration with ³H-inulin, represented about 17% of the leaf blade water content. This volume was used to calculate a global intracellular concentration for each carbohydrate in the leaf blade. Following these simplifying assumptions, the calculated concentration gradient between the leaf's intracellular compartment and phloem sap is nil for sorbitol and could thus allow for the symplastic loading of the phloem of this alditol. However, infiltration of ¹⁴C-labelled source leaves with 2 mMp-chloromercuribenzenesulfonic acid (PC-MBS), a potent inhibitor of the sucrose carrier responsible for phloem loading in sucrose-transporting plants, had a significant effect on the exudation of both labelled sucrose and sorbitol from the phloem. Therefore, in peach, which is a putative symplastic loader according to minor vein anatomy and sorbitol concentration gradients, apoplastic loading may predominate.     

Changes in apoplastic and intracellular leaf sugars induced by the blocking of export in Vicia faba

Sugar export by broadbean ( Vicia faba L. cv. Aguadulce) was blocked by a cold jacket (1 cm-width, 1°C) applied on the petiole of a mature leaf or by heat-girdling the petiole. A time course study was made on the effects of these treatments on apoplastic and intracellular soluble sugars of the leaf in relation to phloem loading and photosynthesis. Blocking of export by heat-girdling induced an inhibition of phloem loading within 10 min, an accumulation of starch within 30 min and a rise in apoplastic sucrose within 60 min. By contrast, apoplastic hexoses and photosynthesis were not affected by this treatment within 8 h and intracellular sugars were not affected within 2 h. The cold jacket also increased the sucrose content of the apoplast. The increase in apoplastic sucrose induced by the cold barrier is reversed upon rewarming and less marked when the sink/source ratio is increased by defoliating all but the leaves studied. The results are discussed in terms of sink/source relationships. They show that the increase in apoplastic sucrose resulting from inhibition of loading is not part of the events leading from blocking of transport to change in carbon partitioning.     

The Effect of Sucrose on Primordium Development and on Protein and RNA Levels in Detached Cotyledons of Sinapis alba L.

Cotyledons of Sinapis alba cultured in the light on filter-papermoistened with water initiated root primordia within 2 daysafter detachment. These increased in size and penetrated thebase of the petiole after a further 3 days. Culture in sucrosesolution in the light inhibited root formation mainly by affectingthe extension phase when many primordia regressed resultingin a decline in the numbers of primordia per petiole. When cotyledonswere cultured in the dark no primordia were initiated in water,but sucrose promoted root formation. Levels of RNA rose to a peak value in the terminal petiole segmentat a time when primordia were being initiated in this region,protein reached a peak 2 days later. This pattern was much lessevident in the subterminal segments. It did not occur at allin cotyledons cultured in sucrose in the light where a smallinitial increase in RNA and protein was followed by a rapiddecline. The level of RNA and protein in the terminal segmentin light-cultured cotyledons in water was related to the numberof light periods given following detachment as was the percentageof cotyledons which formed roots. The effect of sucrose on RNAand protein content was less marked when cotyledons were transferredfrom sucrose to water instead of being maintained in sucrosethroughout. Sucrose clearly modifies the biochemical and structural changestaking place at the petiole base having particularly inhibitoryeffects on the extension phase of the primordia. The evidenceindicates that sucrose could be causing premature ageing ofcells forming the primordia.     

‘3H-testosterone distribution and binding in RAT thymus cells in vivo’

Summary Autoradiography and biochemical investigations showed that [³H]-testosterone where injected intraperitoneally into male white rats was incorporated rapidly into thymus lymphocytes. Thymic cortex contained more silver grains than medulla, and larger lymphocytes were more labelled than medium or small lymphocytes.Cytosol fraction of thymus cells labelledin vivo with [³H]-testosterone, contained the largest quantity of labelled hormone. A 4S cytosol fraction binds [³H]-testosterone. This could be separated by Sephadex chromatography or by linear sucrose gradient centrifugation. Nuclear extract contained also a small quantity of the labelled hormone.     

Sucrose and Glucose Uptake into Beta vulgaris Leaf Tissues : A Case for General (Apoplastic) Retrieval Systems

Concentration curves for sugar and amino acid uptake by Beta vulgaris L. leaf tissues contained both a saturable and a linear component. Similarly shaped curves were obtained for influx of sucrose, glucose, and 3-O-methyl glucose by leaf discs, whole petiole slices, petiole segments containing pith tissue only, and petiole segments containing vascular bundles, although the tissues took up the various sugars via different proportions of saturable versus linear uptake. Two millimolar p-chloromercuribenzenesulfonic acid selectively inhibited the saturable component of sucrose uptake, but had almost no effect on the linear component. Uptake of glucose and 3-O-methyl glucose remained unaffected by p-chloromercuribenzenesulfonic acid treatment. Anoxia was found to inhibit the linear component of both sucrose and 3-O-methyl glucose influx, while the saturable component remained unaffected. The linear component of sucrose uptake was also competitively inhibited by maltose, as well as being selectively promoted by certain exposures to 5 millimolar N-ethylmaleimide, 2 micrograms per milliliter cycloheximide, and high levels of mannitol acting as osmoticum. These results support the proposal that the linear component is due to a process more complex than simple, or exchange, diffusion. It would also appear that the linear transport component utilizes a separate energy source than does the saturable component of sucrose influx.

Evidence for phloem loading from the apoplast was re-examined with respect to the present findings. Saturable sucrose uptake by minor vein tissues may represent retrieval of solute from the free space, which could explain the `apoplastic loading' phenomenon.

     

(14) C]-Assimilate translocation in the light and dark in celery (Apium graveokns) leaves of different ages

The 2 major photosynthetic products and translocated carbohydrates in celery ( Apium graveolens L.) are sucrose and the sugar alcohol, mannitol. Sucrose is produced and utilized in leaves of all ages. Mannitol, however, is synthesized primarily in mature leaves, utilized in young leaves and stored in all leaves. Here we show that mannitol export was lower from young, expanding leaves than from older leaves. After a 10 min pulse of ¹⁴CO₂ and a 2 h chase in the light or dark there was more radioactivity in sucrose than in mannitol in petiole tissues from leaves of all ages. However, after a chase of 15 h in the dark or 6 h in the light followed by 9 h in the dark, mannitol was the predominant [¹⁴C]-labeled carbohydrate remaining in all leaf and petiole tissues. Thus, newly synthesized sucrose was apparently exported at a faster rate than mannitol and more mannitol was partitioned into vacuolar storage pools than was sucrose. It also appears that in the light both sucrose and mannitol were exported, but in the dark, once sucrose pools were depleted, mannitol remained as the predominant substance translocated. Both mannitol and sucrose were unloaded into petiole storage parenchyma tissue, but sucrose was hydrolyzed prior to storage.     

Microautoradiographic study of the incorporation of labelled amino acids into insoluble compounds of the shoot of a higher plant

Summary Radioactive amino acids are fed singly to intact shoots of field pea (Pisum arvense L.) via the transpiration stream. The range of radiosubstrates used is chosen to be representative of those compounds exported in quantity from the root system of field pea. The distribution of radioactivity from each substrate suggests that mature organs actively assimilate materials which enter the shoot through the xylem. During a four hour period 5–20% of the label recovered from mature tissues is present in insoluble form, much of this as protein.A microautoradiographic technique is used to localize insoluble labelled materials in thin (1–2 ) sections of the plant tissues. Certain cell types (xylem parenchyma, cambial initials, mesophyll of leaves, chlorenchyma of stem and petiole, and all elements of the phloem) appear to be particularly active in elaborating protein and other structural constituents from the labelled substrates. Preferential labelling of chloroplasts is observed in cells of mesophyll, while in all cells the wall components appear to be less readily labelled than their contained protoplasts.     

The kinetics of sucrose concentration in the phloem of individual vascular bundles of the Ricinus communis seedling measured by nuclear magnetic resonance microimaging

The sucrose concentration was measured at 70-min intervals in the phloem of individual bundles of the hypocotyl of Ricinus seedlings by ¹H nuclear magnetic resonance (NMR) spectroscopic imaging. The sucrose concentration stayed fairly constant in all bundles for more than 7 h if the cotyledons were embedded in the endosperm or excised and incubated in 100 mM sucrose. If, however, the sucrose solution was replaced by sucrose-free buffer solution, the sucrose levels in the phloem decreased with a kinetic depending on the seedling: in some cases there was a smooth decline, in some a decline followed by a slight recovery and in some cases a clear-cut oscillation. The sucrose concentration was often not identical in the phloem of the individual bundles. The oscillations were larger in the phloem at the apex of the hypocotyl than in the phloem at the base of the hypocotyl. Cutting the petiole of one cotyledon led to a decrease in sucrose not only in the four bundles directly connected to the severed petiole but in all eight bundles of the hypocotyl. Cutting the petiole and dividing the vascular ring at the cotyledonary node and at the root crown did not prevent the decline of sucrose in all eight bundles. Therefore, a functional equilibration of translocated solutes between the eight bundles may occur within the 1-h measuring interval by radial diffusion through the parenchyma of the hypocotyl. Received 4 July 1997 / Accepted: 4 October 1997     

Transport processes in stimulated and non-stimulated leaves ofMimosa pudica

Summary Mature leaves ofMimosa pudica L. or parts of them were exposed to¹⁴CO₂, and translocation was recorded by macroautoradiography. It was observed that considerable amounts of labelled photoassimilates were accumulated in pulvini when the leaf was stimulated. In non-stimulated leaves, no such accumulation of label was observed.Microautoradiographs of pulvinar regions of the non-stimulated leaf showed¹⁴C- label restricted to the phloem. When stimulated, the¹⁴C- label was unloaded from the phloem of the pulvini. Labelled photoassimilates appeared most concentrated in the walls of the collenchymatous cells and beyond in the extensor region of the motor cortex. There, label was accumulated in the apoplastic compartments. Stimulation causes a sudden phloem unloading of sucrose, and its accumulation in the apoplast lowers the water potential which eventually exceeds the osmotic potential of the extensor cells of the motor cortex. By removal of cytoplasmic water the motor cells lose turgidity which results in the closing movement of the leaflets, and — some seconds later — in the bending down of the petiole. In late afternoon night-stimulation triggers sucrose unloading in secondary pulvini. During phases of relaxation, labelled material is taken up by motor cells of the extensor, which concomitantly gain turgor.Part of the doctoral dissertation of Jörg Fromm supported by the Deutsche Forschungsgemeinschaft     

Virus-like particles in phloem tissue of chervil (Anthriscus cerefolium) infected with anthriscus yellows virus

Clusters of isometric virus-like particles c. 22 nm in diameter, embedded in amorphous electron-dense material, were found in the phloem tissue of Anthriscus cerefolium (chervil) plants infected with the semi-persistent aphid-borne virus, anthriscus yellows (AYV). The particles resembled those seen previously in thin sections of AYV-transmitting aphids (Cavariella aegopodii). The particles were found only in the central vascular bundle of the petiole and its continuation in the leaf midrib. They were also found in extracts made by grinding petiole and midrib tissue in 10 % sucrose using Carborundum. These results confirm earlier studies which suggest that AYV is confined to deeper-lying tissues.     

Time course of low temperature inhibition of sucrose translocation in sugar beets

Further studies are presented characterizing the time-course response of sucrose translocation in sugar beet (Beta vulgaris L. cv Klein Wanzleben) to low temperature inhibition. Only the temperature of a 2 cm zone of the source-leaf petiole was varied (1° vs 25°, approximately). The half-time of inhibition, defined as the time required for 50% inhibition of the control or pre-cooling rate, varied from 4 to 15 minutes, and the half-time of recovery from 30 to 100 minutes. Maximum inhibition varied from 68 to 92%. Possible uncertainties in evaluating these parameters are discussed. When the duration of the low temperature period was sufficient to permit essentially full recovery, subsequent re-warming of the petiole zone to 25° to 30° effected little or no increase in the translocation rate. It is evident that the interposition between source and sink of a 2 cm petiole zone maintained at a temperature generally inhibitory to physiological processes resulted in little or no impairment to the translocation process, after a suitable thermal adaptation period. Thermally adapted petiole systems de-adapted after periods as short as 1 hour at 25°.     

Sucrose-Induced Accumulation of beta-Amylase Occurs Concomitant with the Accumulation of Starch and Sporamin in Leaf-Petiole Cuttings of Sweet Potato

β-Amylase of sweet potato (Ipomoea batatas L.), which constitutes about 5% of the total soluble protein of the tuberous root, is absent or is present in only small amounts in organs other than the tuberous roots of the normal, field-grown plants. However, when leaf-petiole cuttings from such plants were supplied with a solution that contained sucrose, the accumulation of β-amylase was induced in both leaf and petiole portions of the explants. The sucrose-induced accumulation of β-amylase in leaf-petiole cuttings occurred concomitant with the accumulation of starch and of sporamin, the most abundant storage protein of the tuberous root. The accumulation of β-amylase, of sporamin and of starch in the petioles showed similar dependence on the concentration of sucrose, and a 6% solution of sucrose gave the highest levels of induction when assayed after 7 days of treatment. The induction of mRNAs for β-amylase and sporamin in the petiole could be detected after 6 hours of treatment with sucrose, and the accumulation of β-amylase and sporamin polypeptides, as well as that of starch, continued for a further 3 weeks. In addition to sucrose, glucose or fructose, but not mannitol or sorbitol, also induced the accumulation of β-amylase and sporamin, suggesting that metabolic effects of sucrose are important in the mechanism of this induction. Treatment of leaf-petiole cuttings with water under continuous light, but not in darkness, also caused the accumulation of small amounts of these components in the petioles, probably as a result of the endogenous supply of sucrose by photosynthesis. These results suggest that the expression of the gene for β-amylase is under metabolic control which is coupled with the expression of sink function of cells in the sweet potato.     

The different growth responses of the Arabidopsis thaliana leaf blade and the petiole during shade avoidance are regulated by photoreceptors and sugar

During the shade-avoidance response, leaf blade expansion is inhibited and petiole elongation is enhanced. In this study, we examined the roles of photoreceptors and sugar on the differential growth of the leaf blade and petiole in shade conditions. Under the conditions examined, cell expansion, not cell division, played a major role in the differential leaf growth. The enhanced cell expansion in the leaf blade is associated with an increase in the ploidy level, whereas cell elongation was stimulated in the petiole in dark conditions without an increase in the ploidy level. Analysis of phytochrome, cryptochrome and phototropin mutants revealed that phytochromes and cryptochromes specifically regulate the contrasting growth patterns of the leaf blade and petiole in shade. Examination of the effects of photo-assimilated sucrose on the growth of the leaf blade and petiole revealed growth-promotional effects of sucrose that are highly dependent on the light conditions. The leaf blades of abscisic acid-deficient and sugar-insensitive mutants did not expand in blue light, but expanded normally in red light. These results suggest that both the regulation of light signals and the modulation of responses to sugar are important in the control of the differential photomorphogenesis of the leaf blade and petiole.     

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.     

Carbon fluxes in mature peach leaves

The turnover and transport of sugars are described in peach (Prunus persica L. Batsch), a species exporting both sucrose and sorbitol. Apparent export rate was slower in peach leaves than in leaves of herbaceous species. Sorbitol was the major soluble end product of photosynthesis and the major soluble carbohydrate in the leaf (higher than sucrose). Carbon fluxes were described using ¹⁴C labeling, radioactivity loss curves, and compartmental analysis during the second half of the photoperiod when chemical steady state was reached for soluble carbohydrates. The measured specific radioactivity of sucrose was typical of a primary product. The delayed decrease in specific radioactivity of sorbitol indicated that part of it was secondarily synthesized. Sucrose is proposed to be the carbon source for the delayed synthesis of sorbitol in the light. The sorbitol to sucrose ratio was higher in the petiole than in the leaf tissues. In phloem sap, obtained using stylectomy of aphids and collected from the main stem between source leaves and apex, this ratio was lower than in the petiole, suggesting a preferential sorbitol demand by sinks.