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.     

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.     

(Questions) on phloem biology. 2. Mass flow, molecular hopping, distribution patterns and macromolecular signalling

This review speculates on correlations between mass flow in sieve tubes and the distribution of photoassimilates and macromolecular signals. Since micro- (low-molecular compounds) and macromolecules are withdrawn from, and released into, the sieve-tube sap at various rates, distribution patterns of these compounds do not strictly obey mass-flow predictions. Due to serial release and retrieval transport steps executed by sieve tube plasma membranes, micromolecules are proposed to “hop” between sieve element/companion cell complexes and phloem parenchyma cells under source-limiting conditions (apoplasmic hopping). Under sink-limiting conditions, micromolecules escape from sieve tubes via pore-plasmodesma units and are temporarily stored. It is speculated that macromolecules “hop” between sieve elements and companion cells using plasmodesmal trafficking mechanisms (symplasmic hopping). We explore how differential tagging may influence distribution patterns of macromolecules and how their bidirectional movement could arise. Effects of exudation techniques on the macromolecular composition of sieve-tube sap are discussed.     

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.     

Characterization of Ricinus communis phloem profilin,RcPRO1

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.     

ONTOGENY AND STRUCTURE OF THE SECONDARY PHLOEM IN PYRUS MALUS

Evert , Ray F. (U. Wisconsin, Madison.) Ontogeny and structure of the secondary phloem in Pyrus malus. Amer. Jour. Bot. 50(1): 8–37. Illus. 1963.—The secondary phloem of apple consists of sieve-tube elements, companion cells, phloem parenchyma cells, fiber-sclereids, and ray parenchyma cells. The sieve-tube elements are generally long, slender cells with very oblique end walls and much-compounded sieve plates. All sieve-tube elements initially possess nacreous thickenings. Similar wall thickenings were observed in the differentiating fiber-sclereids and xylem elements. Of the 245 sieve-tube elements critically examined, 242 were associated with companion cells. All of the companion cells were shorter than their associated sieve-tube elements. Young companion cells possess slime bodies which later become dispersed. Callose is often found on the sieve-tube element side of the common wall between sieve-tube element and companion cell. In several collections, callose was found on both sides of that wall. The parenchyma cells are of 3 types: crystal-containing cells; tannin-and/or starch-containing cells; and those with little or no tannins or starch. Any type parenchyma cell may be on to genetically related to a sieve-tube element, that is, may be derived from the same phloem initial as the sieve-tube element. Morphologically, the phloem parenchyma cells intergrade with the companion cells, the tannin- and starch-free parenchyma cells often being difficult to distinguish from companion cells. Most of the tannin- and starch-free parenchyma cells collapse when the contiguous sieve-tube elements become nonfunctional. The fiber-sclereids arise from parenchyma cells which overwinter on the margin of the cambial zone and differentiate in nonfunctional phloem.     

Localization of Phloem Oxidases

Among oxidases, cytochrome oxidase has been localized in mitochondria of all phloem cells, catalase has been visualized in parenchyma peroxisomes and peroxidase has been localized in cell walls and in several cell organelles. In angiosperms, peroxidase is present in all phloem cell walls; it is sensitive to cyanide inhibition excepted in sieve areas and around plasmodesmata between sieve tubes and companion cells. In some species, this cyanide resistant oxidasic activity can be localized without exogenous H₂O₂. Peroxidase is localized on ribosomes, inside vacuoles, on the tonoplast and often on the plasmalemma in companion cells and differentiating sieve elements. In young sieve cells some dictyosomes can exhibit a strong peroxidasic activity. In mature parenchyma cells peroxidase can be associated with ER cisternae but not with vacuoles.     

Zur Lokalisation von Dehydrogenasen des Energiestoffwechsels im Phloem von Cucurbita pepo L.

Summary In the stem phloem of Cucurbita pepo the enzymes GAPDH, ADH, MDH, NADP-IDH, NAD-IDH, G₆PDH and SDH were localized histochemically with the aid of tetrazolium salt (NBT). When the stems were deep-frozen the most intense formation of formazan was found in companion cells, less in phloem parenchyma cells, and very little in sieve tubes.The distribution of enzymes in phloem markedly changes when stems were cut 2 minutes before freezing: 2,5 cm behind the sectional area little formazan was found. Companion cells and parenchyma cells had lost nearly all activity. 15 cm behind the sectional area there was again a higher concentration of formazan in the companion cells and parenchyma cells. In this region an even higher activity was detected in sieve tubes. 25–30 cm behind the sectional area the distribution of formazan was nearly the same as in the intact stems.Apparently activities of the enzymes tested primaily occur in the companion cells and parenchyma cells only. After wounding they are translocated into sieve tubes or exudate.     

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4–5 m; wall thickness 0.12 m) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 m; wall thickness 0.2 m) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.     

Long-term functioning of enucleate sieve elements: possible mechanisms of damage avoidance and damage repair

Abstract. The sieve-tube elements of long-lived arborescent monocotyledons which lack secondary thickening remain functional for many decades despite lacking a nucleus. A minimal requirement for transport by mass flow powered by the Munch mechanism is maintenance of semi-permeability of the plasmalemma of the sieve tube elements; loading and re-loading could be deputed to nucleate cells symplastically linked to the sieve elements. An additional requirement in the long-term relates to replacement of components damaged by mechanical, chemical or radiation intrusions. Minimizing the damage from radiation and chemical agents can be related to a number of commonly observed features of sieve tubes. Damage from O₂ and radicals derived there from is minimized in these essentially aerobic cells by (1) the absence of intercellular gas spaces in the phloem combined with the lower O₂ solubility and diffusivity in concentrated disaccharide (or sugar alcohol) solutions, (2) the absence of photosynthetic machinery which could generate singlet oxygen, and (3) the presence of at least some components of scavenging mechanisms (glutathione, peroxidase, abscorbate). Non-enzymic glycosylation (exacerbated by O2) of proteins is minimized by the low concentrations of reducing sugars in sieve-tubes. Ultraviolet damage is minimized by UV-absorbing materials between the plant surface and the sieve tubes, including the selerenchymatous cap of fibres on the vascular bundles. The extent to which repair involves symplastic import of polypeptides from nucleate companion cells, thus breaching the −800-Da limit on symplastic transport, is unclear, but it could occur in fully differentiated companion cell-sieve element associations without necessarily upsetting development.     

Solute concentrations of the phloem and parenchyma cells present in squash callus

Abstract. Glutaraldehyde fixation was used to determine the solute concentrations in the various cell types present in tissue cultures of squash ( Cucurbita pepo ). Small pieces of callus were plasmolyzed in a graded series of mannitol solutions and fixed in 20 kg m⁻³ glutaraldehyde adjusted to be isosmotic with the particular plasmolysing solution. The callus samples were further processed using standard electron microscopy techniques. Using this procedure, mature sieve elements that form in squash callus have an osmotic potentional of -2.4MPa. The osmotic potential of the callus sieve elements was comparable to values reported for the sieve tube members of the phloem in intact plants. This ability of callus sieve elements to develop high internal hydrostatic pressures demonstrates that they are capable of phloem loading. However, the osmotic potentials of the surrounding parenchymatous cells and companion cells were only –1.15 and –1.5 MPa, respectively. In contrast to the companion cells of the phloem in intact plant tissues, the osmotic potential of the callus companion cells indicated that they were not directly involved in phloem loading. Several immature sieve elements containing distinct nuclei and vacuoles were observed in the callus granules. These immature sieve elements were plasmolyzed in weaker mannitol solutions (below 0.6kmol m⁻³) than the enucleate sieve elements (1.01 kmol m⁻³ mannitol). The low solute concentrations in immature sieve elements indicated that the ability to load sugars occurs concomitantly with the maturation of the sieve element protoplast.     

SIEVE ELEMENTS IN THE MORPHOLOGICALLY REDUCED MISTLETOE,VISCUM MINIMUM HARVEY (VISCACEAE)

The absence of phloem in some Viscaceae may be a direct result of morphological reduction. Viscum minimum is a highly reduced, South African mistletoe that parasitizes Euphorbia. The aerial portions of this species are 2–3 mm long and consist of only 1–2 intemodes with a terminal triad of flowers. The collateral vascular bundles are composed of tracheary elements, parenchyma, and sieve tubes with associated companion cells. The sieve-tube members are similar to those described from other angiosperms, exhibiting simple sieve plates, P-protein, stacks of ER, plastids, and mitochondria. The morphological reduction of V. minimum thus is not associated with a loss of phloic elements in contrast to other reduced viscaceous mistletoes.     

Anatomical Studies on Secondary Phloem of Euonymus bungeanus

The anatomical structures, especially the type, distribution and arrangement of the constituent elements in the secondary phloem of Euonymus bungeanus Maxim. have been studied. The results showed that the secondary phloem was thicker, consisted of sieve-tube elements, companion ceils ,phloem parenchyma cells ,secretory ceils and rays. Sieve-tube elements, phloem parenchyma cells and secretory cells were alternately arranged in tangential bands, forming a conspicuous zone-like constitution. There was no obvious boundary between the functional phloem and the non-functional phloem. Sieve-tube elements were long, slender cells with very oblique end walls and compound sieve plates. Sieve areas on lateral wall were highly differentiated. Companion cells were triangular in transection and slender in radial section. Mostly,two or three companion cells stayed along with one sieve-tube element. In the functional phloem, phloem parenchyma cells were also slender, containing a few starch grains;but in the nonfunctional phloem they enlarged and contained abundant starch grains. Secretory cells were longer than sieve-tube elements, consisting of rubber-like material. Rays were uniseriate. Finally, the authors also discussed the phylogenetic position of E. bungeanus, which may provide some references for further study of the classification of different genera of Celastraceae.     

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Ultrastructural effects in potato leaves due to antisense-inhibition of the sucrose transporter indicate an apoplasmic mode of phloem loading

To study the export of sugars from leaves and their long-distance transport, sucrose-proton/co-transporter activity of potato was inhibited by antisense repression of StSUT1 under control of either a ubiquitously active (CaMV 35S ) or a companion-cell-specific (rolC) promotor in transgenic plants. Transformants exhibiting reduced levels of the sucrose-transporter mRNA and showing a dramatic reduction in root and tuber growth, were chosen to investigate the ultrastructure of their source leaves. The transformants had a regular leaf anatomy with a single-layered palisade parenchyma, and bicollateral minor veins within the spongy parenchyma. Regardless of the promoter used, source leaves from transformants showed an altered leaf phenotype and a permanent accumulation of assimilates as indicated by the number and size of starch grains, and by the occurrence of lipid-storing oleosomes. Starch accumulated throughout the leaf: in epidermis, mesophyll and, to a smaller degree, in phloem parenchyma cells of minor veins. Oleosomes were observed equally in mesophyll and phloem parenchyma cells. Companion cells were not involved in lipid accmulation and their chloroplasts developed only small starch grains. The similarity of ultrastructural symptoms under both promotors corresponds to, rather than contradicts, the hypothesis that assimilates can move symplasmically from mesophyll, via the bundle sheath, up to the phloem. The microscopical symptoms of a constitutively high sugar level in the transformant leaves were compared with those in wild-type plants after cold-girdling of the petiole. Inhibition of sugar export, both by a reduction of sucrose carriers in the sieve element/companion cell complex (se/cc complex), or further downstream by cold-girdling, equally evokes the accumulation of assimilates in all leaf tissues up to the se/cc complex border. However, microscopy revealed that antisense inhibition of loading produces a persistently high sugar level throughout the leaf, while cold-girdling leads only to local patches containing high levels of sugar. Received: 4 March 1998 / Accepted: 7 April 1998     

The structure of the phloem--still more questions than answers

Long-distance assimilate distribution in higher plants takes place in the enucleate sieve-tube system of the phloem. It is generally accepted that flow of assimilates is driven by an osmotically generated pressure differential, as proposed by Ernst Münch more than 80?years ago. In the period between 1960 and 1980, the pressure flow hypothesis was challenged when electron microscopic images suggested that sieve tubes contain obstructions that would prevent passive flow. This led to the proposal of alternative translocation mechanisms. However, most investigators came to the conclusion that obstructions in the sieve-tube path were due to preparation artifacts. New developments in bioimaging have vastly enhanced our ability to study the phloem. Unexpectedly, in vivo studies challenge the pressure-flow hypothesis once again. In this review we summarize current investigations of phloem structure and function and discuss their impact on our understanding of long-distance transport in the phloem.     

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     

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.     

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.     

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.