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.     

Structures in Sieve Elements Cut with a Cryostat Following Different Rates of Freezing

Vascular bundles of the internodes of squash (Cucurbita pepo)were frozen while still attached to the stem by their ends.Four different methods of freezing were employed. With slowercooling rates the sieve tube contents showed distinct evidenceof damage due to ice-crystal formation while tissues were wellpreserved when rapidly frozen. The sieve tubes contained longitudinallyorientated structures which, in rapidly frozen tissues, apparentlyconsisted of discrete strands which appeared to pass into thesieve plate pores. It is concluded that the method of freezing permits the cuttingof sections which represent the in vivo structure of phloemand the results support the concept of translocation by meansof transcellular strands in sieve tubes.     

The distribution of P-Protein in mature sieve elements of celery

Summary The extent of blocking of sieve-plate pores caused by release of cell turgor was investigated by fixing and processing for electron microscopy a long length of celery (Apium graveolens L.) phloem. Differences in distribution of P-protein within the pores were observed between those cells near the two cut ends, and the central cells.To assess the effect of chemical fixation on the distribution of P-protein, strands of celery phloem (fixed or unfixed, and not treated with cryoprotectants) were frozen in Freon 12 and then freeze-substituted. In sieve elements from unfixed tissue there were a greater number of sieve plates displaying partially open pores.Direct freezing of unprotected phloem tissue in Freon 12 resulted in the formation of ice crystals within the lumen of the sieve elements. Freezing of tissue at rates fast enough to avoid the formation of damaging ice crystals resulted in sieve-plate pores having an unoccluded central channel with a peripheral lining of P-protein. In the lumen of the sieve elements the P-protein filaments occurred as discrete bundles ca. 0.5 m in diameter, and as a parietal layer varying in thickness from 0.1 to 0.5 m.     

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.     

LIGHT MICROSCOPE INVESTIGATION OF SIEVE-ELEMENT ONTOGENY AND STRUCTURE IN ULMUS AMERICANA

At maturity the sieve elements of Ulmus americana L. contain a parietal network of very fine strands of slime which is continuous from one sieve element to the next through the sieve-plate pores. Upon injury this parietal network, which is derived from the slime bodies of immature sieve elements, sometimes becomes distorted into longitudinally oriented strands. Some of these strands frequently extend the length of the cells and often are continuous from one sieve element to the next through the sieve-plate pores. At times past such strands have erroneously been interpreted as normal constituents of the mature sieve-element protoplast. Many mature sieve elements of U. americana contain nuclei, which apparently persist for the life of the sieve elements. In addition, some evidence has been found in mature sieve elements for the presence of a membrane which delimits the parietal layer of cytoplasm, including its network of slime strands, from the vacuolar region of the cell.     

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.     

Plasmatic Filaments and Particles in Mature Sieve Elements of Heracleum sphondylium under the Electron Microscope

By using dimethyl sulphoxide in addition to glutaraldehyde onthe phloem of Heracleum a speedier fixative for phloem sieveelements has been developed, less disruptive to plasmatic filaments(p. f.)- Strands of p. f. under the electron microscope seemto run from sieve element to element, axially through the luminaand the sieve-plate pores. Some of these axial strands giveprofiles which suggest that they exist as tubular structuresin which a series of swellings, 20 to 60 ran in diameter, canbe detected as if microperistalsis were passing along them.Marker particles of five different types were noted, often attachedto wisps of plasmatic filaments. Many of the p. f. seemed tohave a helical substructure.     

The microscopy of P-protein filaments in freeze-etched sieve pores

Intact vascular bundles from Nymphoides peltata (S.G. Gmel.) O. Kuntze, shown to have translocated carbon-14, were freeze-fractured and etched for electron microscopy. The interpretation of freezefractured and etched sieve pores and P-protein filaments seen in them is discussed. The entire widths of most of the sieve pores seen contained filaments separated by less than 100 nm. Their arrangement indicates too high a resistance to flow for pressure flow alone to drive translocation at known rates; pumps would be necessary at places along sieve tubes. However, calculations are presented to show that during the time taken to fix pores, by fast freezing or chemically, the filaments in them could rearrange and move further by Brownian and other motion than the distances between filaments which we need to measure. These calculations show that it is not possible, by microscopy alone, to answer the outstanding question “How are filaments arranged in translocating sieve pores?” with enough certainty to tell us whether pressure flow is adequate to explain translocation where filaments are present. The calculations are relevant also to microscopy of other cell structures which may move.     

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 Existence of Transcellular Strands in Mature Sieve Elements

Light microscope observations provide further evidence of transcellularstrands in sieve elements. Specimens were prepared by a newmethod for stripping sieve tubes from phloem tissue after fixationin aldehyde solutions. Straight-sided membranous structuresappear in sieve elements and are interpreted as the boundarymembranes of transcellular strands. Occasionally it is possibleto follow these structures through a sieve plate. The proposal that transcellular strands are artefacts due todiffraction lines is discussed and, in relation to present results,is proved incorrect. In the interference microscope, colourcontrast is demonstrated between a surrounding matrix and thestrands at a sieve plate; since this colour is a function ofdensity and thickness in the specimen, structures which displaya particular colour must be real. Spherical granules, some of which are more refractive than others,and fibrils, which may occur in parallel groups to form fibrillarstrands, are sometimes seen within straight-sided boundaries,but more often these constituents occur in the lumina of sieveelements. Fibrils may spread out to fill the whole of the sieveelement and fan-like arrays of fibrils are seen issuing fromstrands in sieve elements and phloem exudate. Membranous and fibrillar strands are orientated parallel tothe long axis in fixed, unstained sieve elements. Subsequentstaining reveals an additional constituent in the same sieveelements which is like a typical slime plug and is distinguishablefrom strands by a lack of structure and heavier staining. Autoradiographs of phloem exudate show an association betweenthe distribution of mobile carbon-14 and parts of transcellularstrands. Mobile carbon-14 within strands appears to be orientatedin parallel lines but there is no evidence of a particulatedistribution of the isotope. This result suggests the mobilecarbon-14 followed the distribution of the parallel fibrilsand is not associated with plastids or granules. A pattern of displaced contents in sieve elements, which isrepeated in superposed cells, indicates the presence of resistanceto pressure change along intact sieve tubes. Such resistancewould prevent movement of solution in response to a pressuregradient and is consequently incompatible with the mass-flowtheory. This result can be explained if the movement of vacuolarfluid through sieve pores is blocked by cytoplasm which introducesa resistance to pressure change between cells along a sievetube.     

Strands in Sieve Tubes in Longitudinal Cryostat Sections of Cucurbita pepo Stems

Rapidly frozen vascular bundles of Cucurbita pepo were cut longitudinallyin a cryostat and observed in a Nomarski microscope. Clearlydefined transcellular strands were seen traversing the luminaof sieve elements and passing through the sieve pores. Someof these were 5-8 µm wide and gave the impression of compactand comparatively rigid structures, while others, appearingas ribbons about 3 µm in diameter, seemed to have collapsedand lost their contents during preparation. Many of the thickerstrands were seen to consist of a discrete boundary and parallelsubstructural elements approx. 1 µm in diameter. The resultsare consistent with our previous work and support Thaine's theoryof sieve tube translocation based on a transcellular strandsystem.     

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.     

P PROTEIN IN THE PHLOEM OF CUCURBITA : II. The P Protein of Mature Sieve Elements

During maturation of sieve elements in Cucurbita maxima Duchesne, the P-protein bodies (slime bodies) usually disperse in the tonoplast-free cell. In some sieve elements the P-protein bodies fail to disperse. The occurrence of dispersal or nondispersal of P-protein bodies can be related to the position of the sieve elements in the stem or petiole. In the sieve elements within the vascular bundle the bodies normally disperse; in the extrafascicular sieve elements the bodies often fail to disperse. Extrafascicular sieve elements showing partial dispersal also occur. The appearance of the sieve plate in fixed material is related to the degree of dispersal or nondispersal of the P-protein bodies. In sieve elements in which complete dispersal occurs the sieve plate usually has a substantial deposit of callose, and the sieve-plate pores are filled with P protein. In sieve elements containing nondispersing P-protein bodies the sieve plate bears little or no callose, and its pores usually are essentially "open." The dispersed P-protein components may aggregate into loosely organized "strands," which sometimes extend vertically through the cell and continue through the sieve-plate pores; but they may be oriented otherwise in the cell, even transversely.     

Sieve Elements of Minor Veins in the Leaves of Beta vulgaris L.

End walls of sieve elements of minor veins of the leaves ofBeta vulgaris L. do not contain the multi-perforate sieve plateswhich typically occur on the end walls of sieve-tube membersof major veins. Instead, both end and side walls of the sieveelements of minor veins contain scattered pores which may occursingly or in small numbers. These pores are similar to thosewhich are grouped in sieve plates of major veins in size, possessionof callose and plugs of filaments. In addition to these pores,there are tubular connections 0.1 µ in diameter throughcharacteristically thickened parts of the cell wall betweensieve cells and companion cells. Sieve elements of minor veinsdiffer from those of major veins in structure as well as infunction.     

Distribution of P-protein in Mature Sieve Elements of Cucurbita maxima Seedlings Subjected to Prolonged Darkness

A study has been made of the structure of the sieve tubes inthe phloem of seedlings of Cucurbita maxima kept in total darknessfor 2 or 3 days. All cytoplasmic components were found to beparietal in their distribution. The parietal system was closelyapplied to the cell membrane and appeared to be supported bya continuous framework of endoplasmic reticulum (ER) with whichP-protein was intimately associated. The ER-P-protein complexwas highly compact in some sieve elements and loosened to variousdegrees in others. The pores in the sieve plates were eitherunobstructed or occluded by components of the parietal complexin various ways, occlusion not always being accompanied by noticeabledisruption of the parietal system. In visibly undisturbed sievetubes, in which the ER-P-protein complex was in a highly compactstate, occlusion appeared accidental, arbitrary and withoutany alignment of the components present in the pores. It issuggested that the distribution of the cytoplasmic componentsin the parietal position represents a true-to-life conditionof the sieve tube, preserved due to control of the ‘surge’artefact to which transporting sieve tubes are susceptible.However, the organization of sieve tube probably changes withthe state of transport and the highly compact condition of theER-P-protein complex as well as unobstructed or arbitrarilyobstructed sieve plate pores represent a state of ‘rest’or low transport. Cucurbita maxima, P-protein, sieve elements, phloem, seedlings     

SLIME SUBSTANCE AND STRANDS IN SIEVE ELEMENTS

Studies of the secondary phloem of 6 species of woody dicotyledons revealed that slime is not normally dispersed throughout the vacuole of mature sieve elements, but occurs in the form of discrete strands that traverse the cell and run from cell to cell through the sieve-plate pores. As many as 5 fine strands, each measuring less than 0.5μ in diameter, were observed in a single pore. Less than 30% of pore area was occupied by strands. Thus, the pores are mostly open, and intervacuolar continuity exists between cells. These structural characteristics of pores offer strong support for the concept of mass flow.     

Seasonal Variations of Secondary Phloem Development in Pterocarya Stenoptera and Its Relation to Feeding of Kerria yunnanensis

Early in April of 1987, cells in an undifferentiated state which overwintered on the phloem side of the cambial zone in the branch of Pterocarya stenoptera began to differentiate into merebets of phloem. Cambium divided actively in mid-April and ceased to decide by early-Novembet. Five to eleven bands of fibers alternating with the bands of sieve tubes, companion cells and phloem parenchyma cells produced every year. By mid to late April, new xylem differentiation began. Phloem and xylem differentiation ceased almost simultaneously. Functional sieve tube elements were present all the year round in the phloem. During winter, most sieve tubes produced in the current year ceased functioning, leaving only the zone of functional sieve tube of several rows of cells in width with open pores in the sieve plates. These sieve tubes did not collapse until mid-May. In October, several rows of partially differentiated sieve elements appeared near the cambial zone. They still possessed nuclei. The companion cells had produced but no P-protein. They matured during April of the following year and collapsed by July to September. The life span of sieve elements extended for 8 months at the most. In winter, there were less functional sieve tubes in the branch. This may be one of the reasons that only few Kerria yunnanensis survive on the branch of Pterocarya stenoptera.     

Unplugging the callose plug from sieve pores

The presence of callose in sieve plates has been known for a long time, but how this polysaccharide plug is synthesized has remained unsolved. Two independent laboratories have recently reported the identification of callose synthase 7 (CalS7), also known as glucan synthase-like 7 (GSL7), as the enzyme responsible for callose deposition in sieve plates. Mutant plants defective in this enzyme failed to synthesize callose in developing sieve plates during phloem formation and were unable to accumulate callose in sieve pores in response to stress treatments. The mutant plants developed less open pores per sieve plate and the pores were smaller in diameter. As a result, phloem conductivity was reduced significantly and the mutant plants were shorter and set fewer seeds.Key words: Arabidopsis thaliana, callose, callose synthase, glucan synthase-like, phloem, plasmodesmata, sieve plate     

Electron Microscopic Observations of the Structure of Sieve-connexions in the Phloem of Angiosperms and Gymnosperms

The sieve elements of Pinus silvestris L., Sorbus aucupariaL., Vitis vinifera L., and Cucurbita pepo L. have been examinedelectron microscopically in ultra-thin section, and the structuresof the corresponding sieve areas or sieve plates have been describedand compared. In Pinus the sieve areas contain groups of connectingstrands which enter the wall from the lumen side as individualsbut coalesce within it in the median cavity. This cavity hasdeveloped by wall breakdown in the middle lamella and primarywall region and corresponds to the median nodule visible undera light microscope. Neither in this nor in the other speciesobserved is there any visible closing membrane. Structural differences between the four species are shown tosuggest that the major evolutionary trend in the evolution ofspecialized conducting strands has been the enlargement of theconnecting strands from groups of small separate strands toa smaller number of larger strands as the median cavity becomesenlarged to form a canal through the wall. The connecting strands appear invariably to be dense, highlyosmiophilic and continuous with the cytoplasmic surface of thecell. No signs of micropores or of other tubular structure inthe strands have been found. The structures thus revealed aremore nearly compatible with active transport of materials acrossthe sieve plate than they are with any purely physical mechanism.It is suggested that they are incompatible with any mass flowhypothesis.     

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.