STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATIONS OF THE SIEVE-TUBE MEMBERS

The vascular system of the leaf of Populus deltoides Bartr. ex Marsh, was examined quantitatively, and plasmolytic studies were carried out to determine the solute concentrations of sieve-tube members at various locations in the leaf. Both the total number and total crosssectional area of each cell type decreases with decreasing vein size. Although the proportion of phloem occupied by sieve tubes varies considerably from location to location, a linear relationship exists between cross-sectional area of the vascular bundles and both total and mean cross-sectional area of sieve tubes. Collectively, the cross-sectional area of all tertiary and minor veins feeding into a secondary exceeds the total cross-sectional area of sieve tubes at the base of that secondary. Moreover, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than the total sieve tube volume of the secondary itself. Both tracheary elements and sieve-tube members undergo a reduction in both total and mean crosssectional area in the constricted zone at the base of the leaf. The plasmolytic studies revealed the presence of positive concentration gradients in sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and their associated subjacent midvein bundles and from the upper to lower portions of the median bundle of the midvein.     

STUDIES ON THE LEAF OF AMARANTHUS RETROFLEXUS (AMARANTHACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATION IN THE PHLOEM

The vascular system of the leaf of Amaranthus retroflexus L. was examined quantitatively, and plasmolytic studies were carried out on it to determine the solute concentration in cells of the phloem at various locations in the leaf. The proportion of phloem occupied by sieve tubes varies considerably with vein size and leaf size. Collectively, the cross-sectional area of sieve tubes of all tributaries at their points of entry into either a secondary or midvein far exceeds the total cross-sectional area of sieve tubes at the bases of those major veins. In addition, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than total sieve-tube volume of the secondary vein itself. The plasmolytic studies revealed the presence of positive concentration gradients in the sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and from the tip to the base of the midvein. The C₅₀ (the estimated mannitol concentration plasmolyzing, on the average, 50% of the sieve-tube members) was 1.5 m for minor veins and tips of secondary veins and 1.1 m for the bases of secondaries; 1.3 m for the tip of the midvein and 0.6-0.7 m for the midvein in the basal third of the lamina.     

ANATOMICAL FEATURES OF METAPHLOEM IN STEMS OF SABAL,COCOS AND TWO OTHER PALMS

Sieve tubes in metaphloem of palm stems function throughout the life of the plant and merit close investigation. A stem of Sabal palmetto estimated to be 50 years old was sampled extensively. Variation in length of sieve-tube elements throughout this stem was measured and is discussed. In the metaphloem of individual vascular bundles companion cells are not sharply differentiated from other phloem parenchyma cells. Definitive callose deposits and slime are normally absent from mature sieve tubes, even in fixed material. Otherwise no conspicuous structural features which might account for the longevity of sieve tubes can be discerned. Occlusion of phloem strands after leaf fall is initially by callose deposition on sieve plates followed immediately by tylosoid formation. Similar sampling of Cocos nucifera, Washingtonia robusta and to a lesser extent Archontophoenix alexandrae confirmed these results except for quantitative differences.     

VASCULAR SYSTEM OF LODICULES OF DACTYLIS GLOMERATA L.

The vascular system for the two lodicules in a floret of Dactylis glomerata L. was studied in serial sections. The floret stele contained a few modified tracheary elements and xylem transfer cells enveloped by a phloem of squat sieve-tube members and intermediary cells. A single sieve tube and associated phloem parenchyma exited the right and left sides of the stele and upon nearing the base of each lodicule branched and formed the minor veins of the lodicule. The minor veins underwent limited branching and anastomosing to form a small three-dimensional system which described an arc during its ascent in the adaxial portion of each lodicule. The sieve tubes in the minor veins extended halfway up the lodicule and contained short sieve-tube members with transverse, slightly oblique, or lateral simple sieve plates. The associated phloem parenchyma cells were intermediary cells, companion cells, and less intimate parenchyma cells. Intermediary cells terminated the minor veins and touched the distal ends of the terminal sieve-tube members, which lacked distal sieve plates. Although the transverse area of the sieve-tube members remained constant up the lodicule, the transverse area of the associated phloem parenchyma fluctuated.     

Localization of a protein,immunologically similar to a sucrose-binding protein from developing soybean cotyledons,on the plasma membrane of sieve-tube members of spinach leaves

Immunocytochemical studies using antibodies raised against a 62-kDa membrane protein isolated from developing soybean (Glycine max (L.) Merr.) cotyledons were performed on leaf tissue of spinach (Spinacia oleracea L.). This 62-kDa protein was labeled by 6′-deoxy-6′-(4-azido-2-hydroxy)-benzamidosucrose (HABS), a photoaffinity sucrose analogue (K. G. Ripp et al., 1988, Plant Physiol.88, 1435–1445). Western-blot analysis of spinach plasma-membrane proteins indicated a cross-reactive polypeptide identical in molecular mass to that found in soybean. Indirect immunogold labeling of resin-embedded sections of fully expanded leaf tissue resulted in specific localization of colloidal gold on the sieve-tube plasma membrane. The label was uniform and, except for a few non-specific gold particles over the cell wall, all other cellular organelles and membrane systems were free of label. With the exception of occasional gold particles associated with the companion-cell plasma membrane, all other cell types of the leaf contained little or no label. Control sections treated with non-immune rabbit immunoglobulin-G were also essentially free of label. Immunogold labeling of young leaves, in which the phloem contained no mature sieve-tube members, were free of label for the 62-kDa protein. However, young leaf tissue in which mature or nearly mature sieve tubes could be identified, contained immunolabel associated with the sieve-tube plasma membranes. Similar results were obtained with mature leaf tissue of sugar beet (Beta vulgaris L.). The results of the immunocytochemical studies are consistent with the suggestion that the concentrating step in the phloem-loading process in this species may occur across the sieve-tube plasma membrane. This paper is dedicated to the memory of William A. Dungey     

Proliferating sieve elements present in bud phloem anastomoses connect sieve tubes of axillary bud traces to stelar vascular bundles in the aquatic monocotyledonPotamogeton natans L. (Potamogetonaceae)

Summary The stem ofPotamogeton natans is characterized by a central stelar vascular system with reduced xylem and abundant phloem. Wide sieve tubes composed of short sieve-tube members joined by simple sieve plates and associated with companion cells establish an effective conduit for assimilates. At each node the phloem forms a network of parallel sieve elements connecting the stem phloem to leaf and bud traces. InP. natans an axillary bud rarely develops into a side branch, its procambial vascular bundles are each connected to the nodal complex via separate anastomoses. Their most unusual components are the anastomosai sieve elements (ANSE), characterized by thin cell walls pitted all over by tiny callose-lined pores resembling plasmodesmata, which can be detected as bright areas by fluorescence microscopy after staining with aniline blue. Several layers of ANSE make up the centre of an anastomosis and link to both the nodal and bud stelar sieve tubes via mediating (MSE) and connecting sieve elements (CSE). The ultrastructural differentiation of ANSE, MSE, and CSE corresponds to that of normal sieve elements, i.e., in the mature stage they are enucleate, evacuolate, and have lost most of their cytoplasm. Their plastids are of form-P2c, containing many cuneate protein crystals, typical of monocotyledonous sieve elements. Quantitative aspects of the pore areas are discussed in relation to the functional significance of bud anastomoses.Abbreviations ANSE anastomosai sieve elements - CSE connecting sieve elements - FM fluorescence microscopy - LM light microscopy - MSE mediating sieve elements - TEM transmission electron microscopy Dedicated to Professor Dr. Rainer Kollmann on the occasion of his retirement     

Analysis of the driving forces of phloem transport in Ricinus seedlings: sucrose export and volume flow are determined by the source

The aim of this study was to reveal the factors determining sucrose export and volume flow through the sieve tubes in Ricinus communis L. seedlings. The cotyledons take up sucrose from the apoplasm in vivo, and export most of it to the growing sinks, hypocotyl and root. This simple source-sink system allowed sucrose uptake and export to be studied under controlled conditions with respect to apoplasmic sucrose concentrations. From the additional knowledge of the sucrose concentrations in the mesophyll and the sieve tubes, transmembrane concentration differences were calculated. The volume flow rate along the sieve tubes could be calculated from the export rate and the sucrose concentration in the sieve tubes. While the export rate exhibited saturation kinetics, the volume flow rate decreased at high external sucrose concentrations. The export rate correlated with the sucrose uptake rate, the volume flow rate correlated with the sucrose concentration (osmotic pressure) difference across the sieve-tube plasma membrane, the driving force for transmembrane water flux. From these data it can be concluded that sucrose export and the volume flow through the sieve tubes are determined by activities of the source. Export out of Ricinus cotyledons was considerably higher than export out of green source leaves of different species. The concomitant comparatively low sucrose concentration in the sieve-tube sap of the seedlings can thus be attributed to a very high water flux into and along the sieve tubes associated with the high sucrose flux. Received: 28 November 1997 / Accepted: 4 April 1998     

Electron-Microscopic Evidence of the Vacuolar Nature of Phloem Exudate

We performed electron-microscopic examination of structural diurnal changes in the lumen of sieve tubes and the vacuolar system of corresponding companion cells and changes induced by the experimental blockage of assimilate export from the leaf by its cold-girdling. For these investigations, Cucurbita pepo L. and Helianthus annuus L. plants were used, that is, plant species from groups of symplastic and apoplastic plants, which differ in the type of companion cells and a mode of phloem terminal loading. The examinations showed the complete identity of changes in the electron texture of the sieve-tube lumens and companion-cell vacuoles in both plant species in the course of a day, when the level of assimilates changed, or after export blockage. Similar changes in the structure of the vacuolar labyrinths were stated in the companion cells under normal conditions and after cold-girdling, as related to the rate of sieve-tube loading with the vacuolar exudate. Vacuolar expansion and starch accumulation developing in response to changes in the assimilate level in the evening and after cold blockage of the assimilate export occurred in different types of cells, as dependent on their position in the symplast domains. However, the rate of the process similarly depended on the balance between assimilate synthesis and export. Synchronous changes in the texture of the sieve-tube lumen and companion-cell vacuoles were observed within each complex, but asynchronous changes occurred in different complexes. We suggested this phenomenon for recognizing the particular complexes, when they are grouped in a bundle. We observed no signs of cytoplasm or protein synthetic machinery in the sieve tubes. We concluded that the sieve-tube lumen and vacuoles of companion cells are common in nature. Similar electron texture of the images of the companion-cell vacuolar labyrinth and tube lumens, their connection through the lateral sieve fields, morphological modifications of the companion-cell vacuolar system as dependent on the activity of sieve tube loading—all of these facts imply the continuity of these transport compartments and fluxes in them and the similarity in the composition of the exudates from companion-cell vacuoles and phloem tubes.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): ULTRASTRUCTURE,PLASMODESMATAL FREQUENCY,AND SOLUTE CONCENTRATIONS

The minor veins and contiguous tissues of mature leaves of Populus deltoides Bartr. ex Marsh. were examined with the electron microscope to determine the ultrastructural characteristics of the component cells and to determine the structure, distribution, and frequency of plasmodesmata between the various cell types. In addition, plasmolytic studies were carried out to determine the solute concentrations of the various cell types of the minor veins and contiguous tissues. The cells comprising the mesophyll and bundle sheath contain all the components typical of photosynthetic cells. Paraveinal mesophyll cells and bundle-sheath cells have fewer microbodies and smaller chloroplasts than do palisade parenchyma cells. Vascular parenchyma and companion cells tend to intergrade with one another structurally but can be distinguished from one another by their characteristic plastids. The mature, enucleate sieve-tube member is lined by a parietal layer of cytoplasm consisting of plasmalemma, endoplasmic reticulum, mitochondria, plastids, and P-protein. Plasmodesmata occur along all possible routes from the palisade parenchyma cells to the sieve tubes of the minor veins, and their frequency increases with increasing proximity to the sieve-tube members. Plasmolytic studies revealed that the paraveinal mesophyll cells had a higher C₅₀ (estimated mannitol concentration plasmolyzing, on the average, 50% of a given cell type) than any other cell type of the leaf. Concentration gradients existed along the palisade cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route as well as along the paraveinal mesophyll cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route. Considering the frequency of plasmodesmata along these routes, it is conceivable that photosynthate diffuses from palisade cells to the companion cells along concentration gradients. Within the minor veins, the C₅₀ was higher for sieve-tube members than for either companion cells or vascular parenchyma cells, indicating that loading of the sieve tubes is an active, energy-dependent process.     

A THREE-DIMENSIONAL RECONSTRUCTION OF THE VASCULAR SYSTEM TO THE LODICULES,ANDROECIUM, AND GYNOECIUM OF A FERTILE FLORET OF PANICUM DICHOTOMIFLORUM (GRAMINEAE)

A three-dimensional reconstruction of a fertile floret stele of Panicum dichotomiflorum approaching anthesis was made by a new technique using superimposition of tracings of 80, 1-μm thick serial sections, cleared tracing film, and mounting adhesive. From a collateral bundle, which also served as the median trace to the fertile lemma, most of the vascular tissue branched adaxially and horizontally to become the sole vascular supply to the two lodicules, three stamens, and pistil. The xylem branched at a low level to form a broad and long vessel plexus. The phloem branched at a higher level to overlay the vessel plexus on the right and left with an arc of horizontal sieve tubes in a phloem plexus. Those sieve tubes and vessels which rose after branching from the horizontal plexi assumed a more vertical course in the floret stele. Traces to the right and left lodicules arose from the lower abaxial portions of the flanks of the floret stele. Vessels ascended vertically from the xylem plexus and passed through the phloem plexi and joined with the sieve tubes there to exit at the same level and become the right and left lodicule traces. The vascular tissues to the three filament traces arose from different higher levels of the stele. The sieve tubes for the median filament trace arose vertically from the abaxial side between but above the lodicule traces. At higher levels the sieve tubes for the lateral filaments rose from the horizontal arcs of the flanks of the stele and departed it tangentially. The vessels destined to the filament traces arose in the center of the floret stele from adaxial portions of the horizontal plexus, ascended between the arcs of phloem, exited the stele simultaneously above the phloem of the traces, and followed the courses of their respective sieve tubes. The adaxially displaced apex of the floret stele then contained the vascular tissue related to the pistil. All the sieve tubes and vessels of the floret stele were embedded in a matrix of intermediary cells. The peripheral intermediary cells associated with the vessel plexus were xylem transfer cells with pronounced wall ingrowths. At higher levels in the floret stele, intermediary cells in scattered locations near sieve tubes or vessels had less conspicuous wall ingrowths. No preferred orientation of transfer cells with any particular trace was noted.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

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.     

VASCULAR ARCHITECTURE IN ISOPHYLLOUS AND FACULTATIVELY ANISOPHYLLOUS SPECIES OF PENTADENIA (GESNERIACEAE)

The organization and differentiation of primary vascular tissue in isophyllous shoots of Pentadenia crassicaulis and facultatively anisophyllous shoots of P. orientandina (Gesneriaceae) were compared using serial reconstructions and quantitative methods. Despite clear differences in shoot symmetry, both species are vascularized by four sympodia, with trilacunar, split-lateral nodal anatomy. Leaf trace tracheary element number and diameter reflect leaf size differences in P. orientandina: these parameters are significantly greater in the large ventral leaves than in the small dorsal leaves. The median and lateral traces of ventral leaves of this species have a similar number of tracheary elements of equal diameter, while there are significantly more tracheary elements in the median than lateral traces of dorsal leaves. The pattern seen in P. crassicaulis is similar to that seen in the dorsal leaves of P. orientandina. In both species, protoxylem development anticipates differences in mature shoot vasculature. Changes in tracheary element number during ontogeny precede or are approximately coincident with changes in leaf size. These results suggest that the facultative expression of leaf size differences in P. orientandina is associated with opportunistic development and differentiation of the lateral trace.     

Histochemical localization of nucleoside triphosphatase activity in assimilate conducting plant tissue

Summary For the histochemical localization of nucleoside triphosphatases at the electron microscopic level, prefixed tissues were incubated with lead nitrate in addition to substrate (GOMORI reaction). While ATP and UTP as substrates gave electron-dense reaction products at the plasmalemma of sieve tubes, companion cells and phloem parenchyma cells, and at plasmodesmata in primary pitfields, AMP gave reaction products only at the tonoplast of parenchyma cells. Since electron-dense deposits also occur in cell walls and vacuoles, energy dispersive X-ray microanalysis was used to distinguish between lead deposits and lead-phosphate deposits. The latter were restricted to the symplast. Among the three plant species used, the leaf bundle phloem ofHordeum distichon showed ATPase activity largely restricted to the phloem cells, except for the thickwalled sieve tubes. Some activity also bordered the chloroplasts of the bundle sheath cells. In the C₄ plantGomphrena globosa, ATPase and UTPase activities appeared to be the greater in phloem parenchyma cells than in sieve tubes. In the phloem of youngMonstera deliciosa roots, ATPase occurred not only at the plasmalemma of sieve tubes, but also around sieve-tube plastids. When compared with AMP as substrate, it appears that nucleoside triphosphates are the natural substrates of the enzyme(s) in the plasmalemma of sieve tubes and phloem parenchyma cells.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.     

Structurally,phloem unloading in the maize leaf cannot be symplastic

Developing longitudinal vascular bundles of the leaf blades of maize (Zea mays L. cv. W273) were examined with the transmission electron microscope to determine the frequency of plasmodesmata between the sieve tubes and their neighboring cells. Of particular interest were the protophloem sieve tubes, the first sieve tubes to mature in importing (all large and some intermediate) bundles. The protophloem sieve tubes, most of which lack companion cells, intergrade structurally with the thin-walled metaphloem sieve tubes. Both the protophloem sieve tubes and the thin-walled metaphloem sieve tubes and their companion cells (the sieve tube-companion cell complexes) are virtually isolated symplastically from the rest of the leaf, precluding a symplastic mechanism of phloem unloading in the leaf blade of maize.     

SOLUTE CONCENTRATIONS IN THE PHLOEM AND APEX OF THE ROOT OF ZEA MAYS

Plasmolytic studies utilizing a graded series of mannitol solutions (0.1–1.4 M in 0.1 M increments) were conducted on adventitious roots of Zea mays to determine solute concentrations of cell types at various locations in the root. Results indicated that mature sieve-tube members had the highest solute concentration as determined by their C₅₀ (the estimated mannitol concentration plasmolyzing an average of 50% of a given cell type) of any cell type in the root. In tissue 12 cm from the tip, C₅₀ values calculated for proto- and metaphloem sieve-tube members were 1.15 and 1.19 M, respectively, while in tissue 0.5 cm from the root tip, values for the same cell types were 0.68 and 0.46 M, respectively. The C₅₀ values for sieve elements in tissue 5 cm from the tip were intermediate (1.08 and 1.11 M). Although the companion cells generally plasmolyzed at nearly the same concentrations of mannitol as the sieve elements, their C₅₀ values were slightly lower than adjacent mature sieve elements. The lowest C₅₀ (0.35 M) for any cell type examined was associated with meristematic cells in tissue 0.1 cm from the root tip. Taken collectively, the results indicate that positive concentration gradients exist between mature sieve tubes and meristematic cells of the root apex of maize.     

Cloning of the cDNA for glutaredoxin, an abundant sieve-tube exudate protein from Ricinus communis L. and characterisation of the glutathione-dependent thiol-reduction system in sieve tubes

Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10–30 kDa) present in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individual STEPs in the sieve tubes the anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3′ untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (K _m) for reduced glutathione and cysteine were 2 mM and 50 μM, respectively. Besides l-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations (up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins. Received: 13 December 1996 / Accepted: 28 December 1996     

Studies on the Movement of Solutes between the Sieve Tubes and Surrounding Tissues3: I. INTERFERENCE BETWEEN SOLUTES AND RATE OF TRANSLOCATION MEASUREMENTS

Certain aspects of the secretion of solutes into, and removalfrom, the sieve tubes of isolated stem segments and rooted cuttingsof Salix viminalis have been studied. Sieve-tube sap was obtainedeither as honeydew from whole individuals or via the severedstylets of the aphid Tuberolachnus salignus (Gmelin). It was shown that interference occurred between the chemicallyunrelated solutes, sucrose and the cations potassium and rubidium.On raising the potassium concentration in the sieve-tube sapby passing a solution of this ion through the xylem, the sucroseconcentration declined. When the sucrose concentration fellover a period of days due to respiratory loss of carbohydratesfrom an isolated stem segment, a concomitant rise in eitherthe potassium or rubidium level in the sap occurred. When a solution of sodium was passed through the xylem, theconcentration of this ion in the sieve-tube sap rose, whilstthat of potassium fell at first, but later rose higher thanits initial value, indicating that both antagonism and synergycan occur between these ions. On introducing both these cationsinto the xylem simultaneously, more sodium than potassium wastaken up by the segment, though the increase in the sodium concentrationin the sieve-tube sap was less than that of the potassium. Perfusingthe xylem with a calcium solution had no effect upon the concentrationof potassium in the sieve tube. It has been shown that the rate of translocation of a solutealong the sieve tube, as measured by the two colony technique,depends upon the rate of removal of this solute from the sievetube. The amount of such lateral loss from the sieve tube isrelated to the potential gradient for a solute between the sievetube and surrounding cells.     

Distribution and frequency of plasmodesmata in relation to photoassimilate pathways and phloem loading in the barley leaf

Large, intermediate, and small bundles and contiguous tissues of the leaf blade of Hordeum tvulgare L. ‘Morex’ were examined with the transmission electron microscope to determine their cellular composition and the distribution and frequency of the plasmodesmata between the various cell combinations. Plasmodesmata are abundant at the mesophyll/parenchymatous bundle sheath, parenchymatous bundle sheath/mestome sheath, and mestome sheath/vascular parenchyma cell interfaces. Within the bundles, plasmodesmata are also abundant between vascular parenchyma cells, which occupy most of the interface between the sieve tube-companion cell complexes and the mestome sheath. Other vascular parenchyma cells commonly separate the thick-walled sieve tubes from the sieve tube-companion cell complexes. Plasmodesmatal frequencies between all remaining cell combinations of the vascular tissues are very low, even between the thin-walled sieve tubes and their associated companion cells. Both the sieve tube-companion cell complexes and the thick-walled sieve tubes, which lack companion cells, are virtually isolated symplastically from the rest of the leaf. Data on plamodesmatal frequency between protophloem sieve tubes and other cell types in intermediate and large bundles indicate that they (and their associated companion cells, when present) are also isolated symplastically from the rest of the leaf. Collectively, these data indicate that both phloem loading and unloading in the barley leaf involve apoplastic mechanisms.