Transcellular Strands in Sieve Tubes; What Are They?

We show that sieve elements of Nymphoides peltata (S. G. Gmel.)O. Kuntze contain strands which are bundles of P-protein filaments.We observe the strands under the light microscope (differential-interferencecontrast), and in the scanning electron microscope which showssome of them to be arranged as a parietal network. We find bundlesof filaments which correspond to these strands in sections ofembedded sieve elements in the transmission electron microscope,and also in freeze-fracture replicas of sieve elements in vascularbundles frozen intact while translocating carbon-14. Not allthe strands are necessarily transcellular; some may end in theparietal layer just to the inside of the plasmalemma where theyappear to come in contact with membranes, possibly of endoplasmicreticulum. The filaments in the strands have the same bandedappearance as filaments in the sieve pores. We are unable tofind any membrane or other special boundary round the strands;we propose they should be called ‘filamentous strands’.We suggest that the filaments are aggregated into strands bythe Bernoulli effect when fluid flows through sieve elements.We suggest that the strands may be formed by flow during translocationas well as by flow due to injury.     

Microfilaments in pores between frozen-etched sieve elements

Summary Sieve tubes were frozen before being cut from plants and were prepared for electron microscopy by freeze-etching. Structures that may be interpreted as filaments appeared in and near pores through sieve plates. Their presence suggests that filaments seen in sieve-pores prepared chemically may be there normally. Filaments appeared more numerous and compacted in sieve pores between sieve elements that had been pre-treated with glycerol than in those that had merely been frozen. A sieve element treated with glycerol appeared plasmolysed. No evidence was found for membrane-bound transcellular strands through pores in sieve plates even though membrane-bound transvacuolar strands of cytoplasm appeared clearly in nearby parenchyma cells.     

The Fine Structure of the Sieve Tubes of the Petiole of Nytnphoides peltatum (Gmel.) O. Kunze

A study has been made by electron microscopy of the fine structureof the peti-olar sieve tubes of the water plant Nymphoides peltatum.These are found to have very well-developed nacreous walls.The pores of the sieve plates appear to be filled in functioningsieve tubes with densely staining cytoplasm. The peripheralcytoplasm of the sieve tubes seems to contain an extensive developmentof the endoplasmic reticulum, whose elements become finer nearthe plates and crowd together through the pores. These findingsappear to be compatible with more than one theory of translocation,including the electro-osmotic theory of mass flow.     

Phloem ultrastructure and pressure flow: Sieve-Element-Occlusion-Related agglomerations do not affect translocation

Since the first ultrastructural investigations of sieve tubes in the early 1960s, their structure has been a matter of debate. Because sieve tube structure defines frictional interactions in the tube system, the presence of P protein obstructions shown in many transmission electron micrographs led to a discussion about the mode of phloem transport. At present, it is generally agreed that P protein agglomerations are preparation artifacts due to injury, the lumen of sieve tubes is free of obstructions, and phloem flow is driven by an osmotically generated pressure differential according to Münch's classical hypothesis. Here, we show that the phloem contains a distinctive network of protein filaments. Stable transgenic lines expressing Arabidopsis thaliana Sieve-Element-Occlusion-Related1 (SEOR1)-yellow fluorescent protein fusions show that At SEOR1 meshworks at the margins and clots in the lumen are a general feature of living sieve tubes. Live imaging of phloem flow and flow velocity measurements in individual tubes indicate that At SEOR1 agglomerations do not markedly affect or alter flow. A transmission electron microscopy preparation protocol has been generated showing sieve tube ultrastructure of unprecedented quality. A reconstruction of sieve tube ultrastructure served as basis for tube resistance calculations. The impact of agglomerations on phloem flow is discussed.     

Distorted phytochrome action spectra in green plants

An evaluation was made of the extent which a Münch-type pressure flow mechanism (i.e., osmotically-generated pressure flow) might contribute to phloem transport in soybean. Estimates of sucrose concentrations in source (leaf) and sink (root) sieve tubes were obtained by a negativestaining procedure. Water potential measurements of the leaf and of the nutrient solution allowed calculation of the turgor pressures in source and sink sieve tubes. The turgor difference between source and sink sieve tubes was compared to that required to drive translocation at the observed velocity between the source and sink, as measured by [¹⁴C] photosynthate movement. Sieve-tube conductivity was calculated from the sieve-tube dimensions, assuming an essentially unobstructed pathway. In three experiments, the sucrose concentration was consistently higher in source sieve tubes (an average of 11.5%) than in sink sieve tubes (an average of 5.3%). The ratio of these values (2.3:1) agreed reasonably well with an earlier ratio for source/sink sieve tube concentrations of 1.8:1, obtained by quantitative microautoradiography. The resulting calculated turgor difference (an average of 4.1 bars) was adequate to drive a pressure flow mechanism at the observed translocation velocities (calculated to require a turgor difference of 1.2 to 4.6 bars). No other force need be presumed to be involved.This work was presented in part at a joint U.S.-Australian Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 15–20, 1975; see Fisher (1976)     

Effect of Water Stress on Turgor Differences and C-Assimilate Movement in Phloem of Ecballium elaterium

The effects of water stress on pressure differences and ¹⁴C-assimilate translocation in sieve tubes of squirting cucumber Ecballium elaterium A. Rich were studied. Water stress was induced by transfer of plants from culture solution to a polyethylene glycol 6,000 solution having an osmotic potential of −18.2 atm. Sieve tube turgor, turgor differences between source and sink, and translocation rate were decreased. After 260 minutes of translocation, only 19% of the total fixed ¹⁴CO₂ had moved out of the leaf, compared to the control value of 62% after the same period of time. The results suggest that water stress slows translocation by lowering sieve tube turgor differences, which are essential for the pressure flow mechanism of conduction.     

A Mathematical Treatment of Munch's Pressure-Flow Hypothesis of Phloem Translocation

The steady state solutions of two mathematical models are used to evaluate Münch's pressure-flow hypothesis of phloem translocation. The models assume a continuous active loading and unloading of translocate but differ in the site of loading and unloading and the route of water to the sieve tube. The dimensions of the translocation system taken are the average observed values for sugar beet and are intended to simulate translocation from a mature source leaf to an expanding sink leaf. The volume flow rate of solution along the sieve tube, water flow rate into the sieve tube, hydrostatic pressure, and concentration of sucrose in the sieve tube are obtained from a numerical computer solution of the models. The mass transfer rate, velocity of translocation, and osmotic and hydrostatic pressures are consistent with empirical findings. Owing to the resistance to water flow offered by the lateral membranes, the hydrostatic pressure generated by the osmotic pressure can be considerably less than would be predicted by the solute concentration. These models suggest that translocation at observed rates and velocities can be driven by a water potential difference between the sieve tube and surrounding tissue and are consistent with the pressure-flow hypothesis of translocation.     

Relation of beet yellows virus to the phloem and to movement in the sieve tube

In minor veins of leaves of Beta vulgaris L. (sugar beet) yellows virus particles were found both in parenchyma cells and in mature sieve elements. In parenchyma cells the particles were usually confined to the cytoplasm, that is, they were absent from the vacuoles. In the sieve elements, which at maturity have no vacuoles, the particles were scattered throughout the cell. In dense aggregations the particles tended to assume an orderly arrangement in both parenchyma cells and sieve elements. Most of the sieve elements containing virus particles had mitochondria, plastids, endoplasmic reticulum, and plasma membrane normal for mature sieve elements. Some sieve elements, however, showed evidence of degeneration. Virus particles were present also in the pores of the sieve plates, the plasmodesmata connecting the sieve elements with parenchyma cells, and the plasmodesmata between parenchyma cells. The distribution of the virus particles in the phloem of Beta is compatible with the concept that plant viruses move through the phloem in the sieve tubes and that this movement is a passive transport by mass flow. The observations also indicate that the beet yellows virus moves from cell to cell and in the sieve tube in the form of complete particles, and that this movement may occur through sieve-plate pores in the sieve tube and through plasmodesmata elsewhere.     

Sieve-element differentiation and fluoresceine translocation in wound-phloem of pea roots after complete severance of the stele

Experimental interruption of the root stele of Pisum sativum L. induces in the cortex tissue the development of wound-sieve tubes which bridge the wound and reconnect the vascular stumps. Outside the stele, sieve plates arise from primary pit fields. This origin is confirmed by the distribution of future sieve pores over the original parenchyma cell wall and by remnants of the pitfield cavity in developing sieve plates. Differentiation of wound-sieve elements is similar to that of bundle-sieve elements and includes the chromatolytic disintegration of nuclei as well as the development of typical sieve pores arising from pit-field plasmodesmata. The completion of first woundsieve tubes (indicated by a continuous chain of anilin-blue-positive sieve plates by-passing the wound) was observed 55–62 h after wounding. However, effective translocation, visualized with fluoresceine as a phloem-mobile marker, was not found until 10 h (on average) later. It is suggested that this time delay corresponds to the maturing of the last link within a chain of wound-sieve-tube members. Presumably, enucleate sieve elements with widened pores are a prerequisite for effective phloem translocation.Abbreviations DAPI 4,6-diamidino-2-phenylindole·2 H₂O - ER endoplasmic reticulum Preliminary results of this investigation have been presented at the International Phloem Transport Conference in Asilomar, Cal., USA 1985 (cf. Schulz 1986c)     

Structure of functional soybean sieve elements

Soybean (Glycine max cv. Bragg) petiolar tissue containing translocated ¹⁴C-sucrose was quick frozen, freeze-substituted in acetone or propylene oxide and embedded in Epon. This procedure allowed cytological observations on sieve elements whose functional condition could be verified by microautoradiography. Sieve elements and companion cells were essentially free of ice damage. Aside from a P-protein crystal, the central portion of the sieve tube lumen was devoid of stainable content except in the vicinity of sieve plates. Various sized clumps of stacked endoplasmic reticulum (ER) lined the wall. Superficially, the ER “membranes” seemed to consist of parallel arrays of 100 Å protein fibrils. Although that possibility could not be excluded, it seemed more likely that the fibrils were actually between ER cisternae and that the lipoprotein ER membrane could not be detected readily due to the loss of lipids during tissue preparation. The amount and distribution of proteinaceous material in the vicinity of sieve plates was variable but, when present, still consisted almost entirely of 100 Å fibrils organized into membrane-like arrays. Stacks of ER in various degrees of disorganization and a few 100 Å fibrils were found near sieve plates, with some fibrils extending through the pores. However, most (70%) of the sieve plate pores were essentially free from obstruction. The observations favor an osmotically generated pressure flow mechanism of translocation in soybean.     

The state of the pores in functioning sieve plates

Summary Electron microscopy of Helianthus stem and Saxifraga sarmentosa stolon after first plunging the material into boiling water indicates that the natural condition of the sieve plate pores is to be plugged fairly compactly with slime substance. Electron microscopy of pre-wilted plants demonstrates the same thing. These and other arguments indicate that the slime-filled condition of the pores is not an artifact induced by enzyme action or turgor release, nor is it a consequence of rapid translocation of slime; it is the natural state. This conclusion, very damaging to the pressure-flow hypothesis, is favourable to the electroosmotic theory.     

Mechanism of inhibition of translocation by localized chilling

Arrhenius plots of translocation velocity as a function of petiole temperature show a marked increase in temperature dependence below 10 C in bean (a chilling-sensitive species) but not in sugar beet (chilling-resistant). The increased temperature dependence below 10 C was not observed for cytoplasmic streaming or oxygen uptake in bean. Bean petioles were served to release pressure in order to determine whether sieve tubes are obstructed in cold-treated petioles. The resulting pressure release caused serious displacement of the crystalline protein bodies in the sieve tubes of petioles at 25 C, but in those locally cooled to 0 C for 30 minutes little displacement occurred, indicating obstruction in the latter. An ultrastructural study of sieve tubes in tissue frozen rapidly in situ and dehydrated by freeze substitution revealed that treatment at 0 C for 30 minutes caused structural alteration and displacement of the cytoplasmic material lining the sieve tube wall resulting in occlusion of sieve plates. The sieve plates of the control petioles at 25 C were generally clear of obstructions. The results indicate that inhibition of translocation by chilling in chilling-sensitive plants results from physical blockage of sieve plates rather than from direct inhibition of a metabolic process which drives translocation.     

ULTRASTRUCTURAL FEATURES OF DEVELOPING SIEVE ELEMENTS IN LEMNA MINOR L.—SIEVE PLATE AND LATERAL SIEVE AREAS

Both intact and cut duckweed plants were prepared for electron microscopy. Plants which are prepared intact do not exhibit callose formation during development of sieve-plate pores. Future pore sites can be recognized by the presence of median cavities that are unassociated with callose platelets. These cavities are first seen in the region of the compound middle lamella and are lined by a plasmalemma. As end walls thicken, the cavities increase in size until open pores of uniform width are formed. Mature sieve plates of intact-prepared plants are also devoid of callose. Fully opened pores are lined by a plasmalemma and are only traversed by an occasional tubule of endoplasmic reticulum. Plants which have been cut prior to fixation possess mature sieve plates containing callose. The pores of developing sieve plates in cut plants exhibit small amounts of callose. Except for the lack of callose, lateral wall connections between sieve elements and contiguous cells are similar in development and mature state to those reported for other species.     

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     

TRANSLOCATION OF PHOTOSYNTHATE IN THE BROWN ALGA NEREOCYSTIS

Translocation of photosynthates was found to occur when blades of Nereocystis were illuminated in the presence of C¹⁴ bicarbonates. Rates of translocation averaging 37 cm/hr in the laboratory were observed. Samples from the epidermis, cortex, and medulla of the stipes of plants with blades exposed to tracer showed that the radioactivity in organic compounds was confined to medulla where sieve filaments occurred. Girdle preparations of blades, interrupting the mucilage ducts and leaving the blade medulla intact, allowed translocation to take place. These data indicate that conduction of photosynthate takes place in the medulla. Similarities between the anatomy of algal sieve filaments and angiosperm sieve tubes, coupled with the continuity of the sieve filaments from blade medulla to stipe medulla suggested indirectly that the sieve filaments were conducting elements. Further support of this hypothesis was provided from collections of radioactive exudate from cells in the medulla of the lower stipe that were continuous with the sieve filaments. Tracer applied to the blades was partially recovered as organic material in a clear fluid that collected inside the pneumatocyst. Continued accumulation of radioactivity in this fluid was dependent on living blades; fluid with low specific activity that did not increase during the experiment accumulated slowly if blades were killed with ethanol after an exposure to tracer. It is possible that the system that produced the stipe fluid was part of (or a side effect of) the system responsible for maintaining volume in the conducting system. It may also provide an alternate route (other than the sieve filaments) for delivery of photosynthates to the base of the plant. Carbon-14 applied to blade tips as bicarbonate was recovered in part as radioactive mannitol in the translocation stream.     

TUBULAR AND FIBRILLAR COMPONENTS OF MATURE AND DIFFERENTIATING SIEVE ELEMENTS

An ontogenetic study of the sieve element protoplast of Nicotiana tabacum L. by light and electron microscopy has shown that the P-protein component (slime) arises as small groups of tubules in the cytoplasm. These subsequently enlarge to form comparatively large compact masses of 231 ± 2.5 (SE)A (n = 121) tubules, the P-protein bodies. During subsequent differentiation of the sieve element, the P-protein body disaggregates and the tubules become dispersed throughout the cell. This disaggregation occurs at about the same stage of differentiation of the sieve elements as the breakdown of the tonoplast and nucleus. Later, the tubules of P-protein are reorganized into smaller striated 149 ± 4.5 (SE)A (n = 43) fibrils which are characteristic of the mature sieve elements. The tubular P-protein component has been designated P1-protein and the striated fibrillar component P2-protein. In fixed material, the sieve-plate pores of mature sieve elements are filled with proteinaceous material which frays out into the cytoplasm as striated fibrils of P2-protein. Our observations are compatible with the view that the contents of contiguous mature sieve elements, including the P-protein, are continuous through the sieve-plate pores and that fixing solutions denature the proteins in the pores. They are converted into the electron-opaque material filling the pores.     

Distribution of leaf assimilates in the stem of Picea abies L.

Summary Autoradiographic and microautoradiographic studies of 2-year-old Picea abies plants show that in summer leaf assimilates from the second-year shoot are translocated basipetally. Leaf assimilates are first transported to the stem via leaf trace phloem, then to the base of the stem in the sieve cells of the latest increment of secondary phloem. On the way down leaf assimilates move radially from sieve cells into cells of the phloem parenchyma, the vascular cambium, the rays, the inner periderm and certain cells of pith and cortex, including the epithelial cells surrounding the resin ducts. Other cells of pith and cortex remain nearly free of label, despite the long translocation time (20 h). With the exception of the vascular cambial cells, the stem cells that gain leaf assimilates by radial distribution coincide with those that contain chlorophyll and starch.     

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system.Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy.Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates.Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.     

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade‐off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier–Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.     

SOME FINE STRUCTURAL OBSERVATIONS ON DEVELOPING AND MATURE SIEVE ELEMENTS IN THE BROWN ALGA LAMINARIA SACCHARINA

The differentiation of sieve elements from inner cortical cells of the stipe of Laminaria saccharina (L.) Lamour. involves the development of a well-structured protoplast and an end wall possessing evenly spaced pores which are visualized by electron microscopy. The protoplast consists of organelles which are commonly found in brown algal cells, including nuclei, cup- or horseshoe-shaped chloroplasts, dictyosomes, mitochondria, and ER. Mitochondria and clusters of small vacuoles, presumably redistributed by the surging effect which occurs in sieve elements, were routinely observed in the vicinity of the end wall. Chloroplasts were seen in progressively degenerated states in older sieve elements, yet nuclei were determined to be non-necrotic. Numerous pores along the end walls interconnect adjacent sieve elements. Each pore is traversed by a strand of cytoplasm and surrounded by plasmalemma. The pores are open and possess no callose. In this paper the sieve element ultrastructures of L. saccharina are compared to those in L. groenlandica, Alaria marginata, Nereocystis lutkeana and Macrocystis pyrifera, and a possible phylogenetic specialization of sieve elements is presented in table form and discussed.