Uptake and transfer of nutrients in ectomycorrhizal associations: interactions between photosynthesis and phosphate nutrition.

Energy-dispersive X-ray microanalytical investigations and microautoradiographic studies were carried out to examine whether the uptake and transfer of phosphate (P) by an ectomycorrhizal fungus is affected by the carbohydrate supply of its host plant. For this purpose, non-mycorrhizal seedlings of Pinus sylvestris L. and plants inoculated with the ectomycorrhizal basidiomycete Suillus bovinus (L. ex Fr.) Kuntze were placed in the dark for 7 days in advance of a P supply. The subcellular element distribution and the uptake and distribution of (33)P was analyzed in non-mycorrhizal and mycorrhizal roots of these plants and compared with plants kept constantly under normal light conditions (control plants). The results show that placing non-mycorrhizal plants in the dark in advance of the nutrient supply led to (1) a reduction of the subcellular contents of P, S and K, but to an increase in the cytoplasmic Na content, and (2) an increase of (33)P absorption and translocation to the shoot. It can be assumed that this increased inflow of (33)P in non-mycorrhizal plants was due to P starvation after suppressed photosynthesis and reduced respiration of these plants. The suppression of photosynthesis by an ectomycorrhizal host plant and the resulting lower carbohydrate supply conditions for the ectomycorrhizal fungus led to (1) a decrease of P absorption by the mycobiont, (2) a change of the P allocation in the fungal cell compartments of an ectomycorrhizal root, and (3) a reduction of P transfer to the host plant. However, microautoradiographic studies revealed that, under these conditions, P was also absorbed by the mycorrhizal fungus and translocated via the Hartig net to the host plant. In mycorrhizal roots of plants placed in the dark in advance of the nutrient supply, the cytoplasmic P content of the Hartig net was reduced and, instead, a high number of polyphosphate granules could be detected within the hyphae. The results indicate that the exchange processes between the symbionts in a mycorrhiza are possibly linked and that P uptake and translocation by an ectomycorrhizal fungus is also regulated by the carbohydrate supply of its host plant.

Microautoradiographic localization of phosphate and carbohydrates in mycorrhizal roots of Populus tremula x Populus alba and the implications for transfer processes in ectomycorrhizal associations.

Microautoradiographic studies were carried out to examine the distribution and exchange of phosphate and labeled carbohydrates in mycorrhizal roots of Populus tremula x Populus alba L. following application of 33P-orthophosphate (Pi) and 14CO2. Labeled Pi was not homogeneously distributed along the mycorrhizal longitudinal axis. The fungal sheath and the Hartig net contained more 33Pi in the median parts of the root than in the apical or basal root zones, indicating that uptake and transfer of Pi to the host plant was localized mainly in this area. The Pi was translocated by the Hartig net and the interfacial apoplast to the host plant. It was distributed by way of the stele within the plant. Young leaves and meristematic tissue in the shoot tip were the main sinks for Pi. In plants that were left in the dark for 5 days before 33Pi application, the reduced carbohydrate supply caused a decrease in Pi absorption by mycorrhizal roots. Microautoradiography of mycorrhizal roots after assimilation of 14CO2 revealed that: (1) the fungal partner had a high capacity to attract photosynthates; (2) the main transfer of carbohydrates was localized in the median zone of a mycorrhizal root; (3) carbohydrates that were absorbed by the mycorrhizal fungus were translocated to the fungal sheath and were homogeneously distributed; and (4) in the main exchange zone, cortical cell nuclei showed a high sink capacity, indicating increased metabolic activity in these cells. We postulate that (1) the phosphate demand of the host plant regulates absorption of Pi by the fungus, and (2) a bidirectional transfer of carbohydrates and Pi occurs across the same interface structure in ectomycorrhizal roots of Populus.

Microautoradiographic studies of phloem loading and transport in the leaf of Zea mays L.

Microautoradiographs showed that [(14)C]sucrose taken up in the xylem of small and intermediate (longitudinal) vascular bundles of Zea mays leaf strips was quickly accumulated by vascular parenchyma cells abutting the vessels. The first sieve tubes to exhibit (14)C-labeling during the [(14)C]sucrose experiments were thick-walled sieve tubes contiguous to the more heavily labeled vascular parenchyma cells. (These two cell types typically have numerous plasmodesmatal connections.) With increasing [(14)C]sucrose feeding periods, greater proportions of thick- and thin-walled sieve tubes became labeled, but few of the labeled thin-walled sieve tubes were associated with labeled companion cells. (Only the thin-walled sieve tubes are associated with companion cells.) When portions of leaf strips were exposed to (14)CO2 for 5 min, the vascular parenchyma cells-regardless of their location in relation to the vessels or sieve tubes-were the most consistently labeled cells of small and intermediate bundles, and label ((14)C-photosynthate) appeared in a greater proportion of thin-walled sieve tubes than thick-walled sieve tubes. After a 5-min chase with (12)CO2, the thin-walled sieve tubes were more heavily labeled than any other cell type of the leaf. After a 10-min chase with (12)CO2, the thin-walled sieve tubes were even more heavily labeled. The companion cells generally were less heavily labeled than their associated thin-walled sieve tubes. Although all of the thick-walled sieve tubes were labeled in portions of leaf strips fed (14)CO2 for 5 min and given a 10-min (12)CO2 chase, only five of 72 vascular bundles below the (14)CO2-exposed portions contained labeled thick-walled sieve tubes. Moreover, the few labeled thick-walledsieve tubes of the "transport region" always abutted (14)C-labeled vascular parenchyma cells. The results of this study indicate that (1) the vascular parenchyma cells are able to retrieve at least sucrose from the vessels and transfer it to the thick-walled sieve tubes, (2) the thick-walled sieve tubes are not involved in long-distance transport, and (3) the thin-walled sieve tubes are capable themselves of accumulating sucrose and photosynthates from the apoplast, without the companion cells serving as intermediary cells.

Sucrose in the free space of translocating maize leaf bundles.

Following exposure of portions of mature maize (Zea mays L.) leaf strips to (14)CO(2), xylem exudate from the leaf strips contained [(14)C]sucrose. Sucrose was the only sugar in the xylem exudate which was obtained from the cut surface of the leaf strips by reducing the external pressure. The sucrose found in the xylem exudate apparently was obtained from the free space of the vascular bundles, its concentration amounting up to 0.25%. When [(14)C]glucose or [(14)C]fructose was supplied in the dark to one end of a maize leaf strip, each was taken up by the xylem, and transported to the opposite end. Xylem exudate from such leaf strips contained (14)C-labeled sucrose in addition to the (14)C-labeled hexose. The results of this study support the view that sucrose is loaded into the companion cell-sieve tube complexes from the apoplast of the vascular bundles in the maize leaf.

Leaf structure in relation to solute transport and phloem loading in Zea mays L.

Small and intermediate (longitudinal) vascular bundles of the Zea mays leaf are surrounded by chlorenchymatous bundle sheaths and consist of one or two vessels, variable numbers of vascular parenchyma cells, and two or more sieve tubes some of which are associated with companion cells. Sieve tubes not associated with companion cells have relatively thick walls and commonly are in direct contact with the vessels. The thick-walled sieve tubes have abundant cytoplasmic connections with contiguous vascular parenchyma cells; in contrast, connections between vascular parenchyma cells and thin-walled sieve tubes are rare. Connections are abundant, however, between the thin-walled sieve tubes and their companion cells; the latter have few connections with the vascular parenchyma cells. Plasmolytic studies on leaves of plants taken directly from lighted growth chambers gave osmotic potential values of about-18 bars for the companion cells and thin-walled sieve tubes (the companion cell-sieve tube complexes) and about-11 bars for the vascular parenchyma cells. Judging from the distribution of connections between various cell types of the vascular bundles and from the osmotic potential values of those cell types, it appears that sugar is actively accumulated from the apoplast by the companion cell-sieve tube complex, probably across the plasmalemma of the companion cell. The thick-walled sieve tubes, with their close spatial association with the vessels and possession of plasmalemma tubules, may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream. The transverse veins have chlorenchymatous bundle sheaths and commonly contain a single vessel and sieve tube. Parenchymatic elements may or may not be present. Like the thick-walled sieve tubes of the longitudinal bundles, the sieve tubes of the transverse veins have plasmalemma tubules, indicating that they too may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream.

Distribution and structure of the plasmodesmata in mesophyll and bundle-sheath cells of Zea mays L.

In leaf blades of Zea mays L. plasmodesmata between mesophyll cells are aggregated in numerous thickened portions of the walls. The plasmodesmata are unbranched and all are characterized by the presence of electron-dense structures, called sphincters by us, near both ends of the plasmodesmatal canal. The sphincters surround the desmotubule and occlude the cytoplasmic annulus where they occur. Plasmodesmata between mesophyll and bundle-sheath cells are aggregated in primary pit-fields and are constricted by a wide suberin lamella on the sheath-cell side of the wall. Each plasmodesma contains a sphincter on the mesophyll-cell side of the wall. The outer tangential and radial walls of the sheath cells exhibit a continuous suberin lamella. However, on the inner tangential wall only the sites of plasmodesmatal aggregates are consistently suberized. Apparently the movement of photosynthetic intermediates between mesophyll and sheath cells is restricted largely or entirely to the plasmodesmata (symplastic pathway) and transpirational water movement to the cell walls (apoplastic pathway).

The influence of externally supplied sucrose on phloem transport in the maize leaf strip.

Sucrose (2,5-1000 mmol l(-1)), labeled with [(14)C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Zea mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted Hoagland's nutrient solution. When the Hoagland's solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of (14)C-labeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of (14)C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of (14)-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 25 mmol l(-1)) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 mmol l(-1) sucrose was the sink effect of the darnened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue more rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink.

The influence of externally applied organic substances on phloem translocation in detached maize leaves.

Solutions of organic substances show differing influences on the direction of phloem transport of (14)C-labeled assimilates in predarkened maize leaf strips, when externally applied to one end of the strip. One group of substances "pushes" the assimilates away from the site of application. Examples of this group are 75 mM solutions of sucrose, trehalose, maltose, D-glucose, D-fructose, glucose-6-phosphate, raffinose and galactose. There is strong evidence that "pushing" substances are taken up from the apoplast and loaded into the phloem. Another group of substances attracts the assimilates, it seems to "pull" the assimilates in direction to the site of application. Examples of this second group are 75 mM solutions of arabinose, melibiose, myo-inositol, D-mannitol, polyethylene glycol 2000, and Na2-EDTA (ethylene-diaminetetraacetate). The "pulling" substances obviously are not taken up into living cells. It is assumed that they accumulate in the apoplast and build up a water stress (water potential), which is counteracted by an increase of solute concentration in the parenchyma, thus creating a sink for assimilates. A third group of substances shows inert behaviour, having no perceptible influence on phloem transport, at least not, when applied as 75 mM solutions. At concentrations of more than 300 mM, inert substances tend to attract assimilates like those of the second group. Inert substances are xylose, sorbose, 2-deoxy-D-glucose, mannose and sorbitol.

The influence of light, darkness, and lack of CO2 on phloem translocation in detached maize leaves.

Longitudinal strips from leaf blades of Zea mays L., with veins continuous along their whole length, proved to be a very uniform and convenient material for translocation experiments. Under normal photosynthetic conditions a very strong basipetal assimilate movement was shown. In the dark this movement persisted as long as starch reserves were available. Parts of the strips exposed to darkness or to CO2-free air, i.e. nonphotosynthetic conditions, became strong sinks which attracted assimilates, darkness having the strongest effect. Microradioautographs showed that transport of assimilates took place in the sieve tubes of the phloem.

Proteins of the sieve-tube exudate of Cucurbita maxima.

Sieve-tube exudate which appears on cut surfaces of stems of Cucurbita maxima as distinct droplets has been depicted in electron micrographs of longitudinal sections of the phloem. The exudate, which was produced from mature sieve tubes only, contained filaments of P-protein, but no mitochondria or vesicles of endoplasmic reticulum. The water-soluble part of the exudate contained at least 12 proteins, as shown by disc-electrophoresis. Enzymic activity was found for peroxidases, acid phosphatases, and aldolases. Color tests and assays for other enzymes, including ATPase, fructokinase, several dehydrogenases, and UDP-glucose: D-fructose-2-glucosyl transferase, gave negative results. With repeated cutting of a stem, the protein content of the exudate increased, while the amount of exudate decreased.

Translocation in Perennial Monocotyledons Tradescantia plants are still functional.

Gross autoradiography, historadiography, and electron microscopy provide evidence that enucleate sieve elements in basal internodes of relatively old Tradescantia plants are still functional.