Development of the Sieve Plate in Saxifraga sarmentosa L.

The differentiating sieve plate in the phloem of the stolonof Saxifraga sarmentosa L. was studied with the electron microscope.Development of the pore site begins with differentiation ofa pair of collar-like areas around the plasmodesma which canbe seen in the youngest identifiable sieve plates. Further growthof the collars occurs by deposition of an amorphous substance,presumably caflose. Although the growth of the collars is simultaneouswith the growth of the surrounding cell wall it is rapid atfirst and the pore sites appear asdome-shaped protuberances.It also involves deposition of callose over an increasinglywider area of the cell wall and since the thickening of thenormal cell wall continues only where notcovered by callose,the collars assume a conical form. There seems to be no displacementor lysis of normal cell wall material during growth of the collars.Eventually the growth of the cell wall in thickness overtakesthe pore sites so that when the growth of the cell wall is completethe pore sites appear as depressions in the sieve plate. Theperforation of a pore site is accomplished by widening of theplasmodesmatal cylinder which begins at the middle lamella byremoval of callose. Endoplasmic reticulum is found in closeproximity to the plasmodesma andis believed to penetrate it.     

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

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

Symplasmic connections between sieve element and companion cell in the stem phloem ofVicia faba L. have a molecular exclusion limit of at least 10 kDa

High-molecular-weight fluorochromes were intracellularly injected into a sieve element of the fascicular stem phloem ofVicia faba L., using a modified membrane-potential-recording pressure probe. After stabilization of the membrane potential following microelectrode impalement, either LYCH (Lucifer Yellow CH), 4.4-kDa FITC-dextran (fluoresceinisothiocyanate-dextran) conjugate, or 3-kDa, 10-kDa or 40-kDa LYCH-dextran conjugate was microinjected into the sieve element. Longitudinal fluorochrome movement across the sieve plates and lateral displacement to the companion cells was detected with all the probes except the 40-kDa conjugate. This indicates that the molecular exclusion limit of the pore/plasmodesma units between a sieve element and a companion cell in the fascicular stem phloem ofVicia faba lies between 10 kDa and 40 kDa.Abbreviations FITC fluoresceinisothiocyanate - LYCH Lucifer Yellow CH - MEL molecular exclusion limit - PPU pore/plasmodesma unit - SE/CC-complex sieve element/companion cell complex     

Differentiation of the Sieve Plate of Cucurbita: A Further View

A re-examination of sieve plate differentiation in Cucurbitamaxima was undertaken in order to determine the relation betweenthe development and dissolution of pore sites and the growthof the permanent part of a sieve plate. Pieces of young petiolesand internodes fixed in glutaraldehyde and post-fixed in osmiumwere used for the study. A pore site first becomes destinguishableby a pair of flat callose collars around a plasmodesma, oneon either side of the sieve plate. The enlargement of the collarsis rapid and the delimitation of the pore sites is more or lesscomplete before any significant thickening has occurred in thesieve plate elsewhere. Later, however, the thickening of therest of the wall overtakes the height of the collars so thateventually the pore sites appear as depressions. The early wallsandwiched between the callose collars remains distinct throughoutpore development. The process of perforation seems to involvea more or less simultaneous disappearance of the callose collarsand the sandwiched layer. Cisternae of endoplasmic reticulumassociated with the pore site seem to be connected with theplasmodesmatal core at all stages of differentiation which addssupport to the view that the endoplasmic reticulum plays anactive role in the process of pore formation.     

SPIRAL TUBULES IN PALM PHLOEM

Spiral tubule structures were observed in sieve elements of Pritchardia and Cocos palms. The spiral tubules were 80–120 nm wide and composed of alternating electron-lucent and electron-dense bands 11–16 nm wide which spiraled around a central core.     

Age-related and origin-related control of the numbers of plasmodesmata in cell walls of developing Azolla roots

Plasmodesmata were counted in the longitudinal and transverse walls in developmental sequences of merophytes in roots of Azolla pinnata R.Br. The differences between certain categories of longitudinal wall were traced to factors that govern the surface area of the cell plates, the density of plasmodesmata (number per unit area of cell plate), and the amount by which each type of plate expands. No evidence for secondary augmentation of plasmodesmatal numbers after the cell-plate stage of development was found, but plasmodesmata are lost from the walls of sieve and xylem elements during their differentiation. Losses caused by cell separation occur in other tissues. The relatively high density of plasmodesmata in transverse walls is based not so much on a high density in the cell plates as on the relatively low expansion that these walls undergo. There appears to be a compensatory mechanism that relates initial plasmodesmatal density to the future expansion of the cell plate. The root shows determinate growth, the apical cell dividing about 55 times. Beginning at about the 35th division there is a progressive failure to maintain the plasmodesmatal frequencies that were developed in earlier cell divisions in the apical cell. The divisions that occur within the later-produced merophytes also show progressive diminution of plasmodesmatal numbers. The result is that the apex of the root, and particularly the apical cell, becomes more and more isolated symplastically, a phenomenon which could account for its limited lifespan and the determinate growth pattern of the root.     

Characteristics of Structure and Differentiation in the Sieve Element of Lower Vascular Plants

The structure and differentiation of the sieve element of lower vascular plants is reviewed using data obtained primarily from ultrastructural investigations conducted during the last ten years. During the last decade the phloem of representatives from every major group of the ferns and fern allies has been examined with the electron microscope and from these studies a rather clear picture has emerged of the structure of the sieve element protoplast in this diverse group of plants. Present data indicate that although the details of sieve-element differentiation may differ, the protoplasts of the mature sieve elements in the various groups of lower vascular plants are remarkably similar in structure. Each consists of a plasmalemma, a parietal, anastomosing network of smooth ER, plastids, mitochondria and, with the exception of the lycopods, variable numbers of refractive spherules. The protoplasts of mature sieve elements are joined by plasmalemma-lined connections, each arising from a single plasmodesma during the course of sieve element differentiation. The size of the connections in the mature elements range from plasmodesmata-like structures to relatively wide sieve-area pores, depending on the species. Moreover, the contents of the cytoplasmic connections vary somewhat according to the species. Whereas in the lycopods, the sieve-area pores are virtually unoccluded by any cytoplasmic material, the cytoplasmic connections of all other lower vascular plants examined with the electron microscope contain variable amounts of membranous material, apparently tubular elements of ER. In Equi-setum hyemale, Psilotum nudum and the eusporangiate and protoleptosporangiate ferns, the ER membranes are very numerous and virtually occlude the pores. Furthermore, the membranes apparently are not connected with the parietal ER in the lumen of the cell. The sieve-area pores of the leptosporangiate ferns also contain ER membranes, however, they are not as abundant as the membranes of the eusporangiate and protoleptosporangiate ferns. In addition, in the leptosporangiate ferns the pore membranes apparently are united with the parietal ER in the lumen of the cell.     

A new interpretation of plasmodesmatal ultrastructure

Summary It is shown that simple, unbranched, plasmodesmata between young xylem ray cells of willow have no direct intercellular continuity apart from the plasmalemma which limits the cytoplasm and lines the plasmodesmatal canal. Each plasmodesma is traversed by a 200 Å diameter tubule (the desmotubule) which has a wall with probably 11 subunits arranged around a central cavity through which runs a 40 Å diameter rod. This rod is connected to the inside of the tubule wall, by fine filaments. At the ends of each plasmodesma the plasmalemma and cell wall are closely appressed to the tubule, thus precluding direct continuity between the cytoplasm of adjacent cells. Through the central part of the plasmodesmata the tubule is separated from the plasmalemma by a 90–100 Å wide gap. Cytoplasmic microtubules in the same tissue have a diameter of approximately 250 Å and a wall probably composed of 13 subunits: both desmotubules and cytoplasmic microtubules therefore have a centre-to-centre subunit spacing of about 47 Å. It is suggested that the desmotubules are not microtubules but may be nuclear spindle fibres which become trapped in the wall during cell plate formation. The endoplasmic reticulum, while closely approaching the plasmodesmata, is not continuous across them. It is thought most unlikely that the endoplasmic reticulum traverses plasmodesmata, as the dimensions of the central tubule — found here as well as by other workers — are smaller than those which would be expected to allow a stable molecular configuration in a unit membrane. The plasmalemma, where it lines the plasmodesmatal canal, appears to have particulate subunits in the outer opaque layers and the presence of these subunits may be attributable to the need for stability in membranes arranged about so small a radius.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Modified pore canals in the cuticle of Gammarus (Crustacea : Amphipoda); A study by scanning and transmission electron microscopy

A novel type of pore canal is described from the cuticle of three species of Gammarus. Each canal passes from the epidermis vertically through the endocuticle and exocuticle, and in the most distal layers of the latter is slightly expanded. Before entering the epicuticle the canal narrows, forming a neck the base of which is encircled by an electron-dense collar. Several tubular structures arise from the collar and pass distally into the reticular innermost regions of the epicuticle. Within the neck and just below its opening at the cuticle surface, a rod-like structure is inserted; this protrudes a short distance from the pore. Each pore canal is connected to many necks; the openings of the latter are aligned in rows over the surface, the openings and rows being about 0.15 and 1.0 μm apart, respectively. Changes in the pore and canal contents are visible and their significance is discussed.     

ULTRASTRUCTURE OF THE FEEDING APPARATUS AND MYONEMAL SYSTEM OF THE HETEROTROPHIC DINOFLAGELLATE PROTOPERIDINIUM SPINULOSUM1

The feeding veil or pallium of the thecate heterotrophic dinoflagellate Protoperidinium spinulosum Schiller is a highly vesiculate membranous sac containing several arched, sometimes bifurcated microtubular ribbons. It originates from an internal microtubular basket, passes through a sphincter-like osmiophilic ring located inside the posterior flagellar pore, and emerges from the cell at that pore. The osmiophilic ring is part of an interconnected myonemal system (composed of two striated collars and several striated connectives) that is anchored to the pore plate and to two inward protrusions composed of minute sulcal plates. A related species, Protoperidinium punctulatum (Paulsen) Balech, also possesses a microtubular basket/osmiophilic ring complex. Elongate electron-dense bodies within the basket resemble digestive secretory granules found in other protists. Granular, electron-lucent microbodies clustered at the anterior end of the basket may also have a role in prey digestion. Dense membranous whorls observed within a P. spinulosum cell presented as it was preparing to initiate feeding indicate a condensed storage site for pallium membranes. A narrow microtubule-strengthened pseudopodal appendage found in two non-feeding cells constitutes the tow filament that serves as the initial linkage between the dinoflagellate and its food. The structures that constitute the pallium and pallium precursors, described here for the first time, are unlike those of other known protists, although some similarities with the dinoflagellate peduncle are evident. The existence of this unique system of organelles may have important ramifications in the search for evolutionary relationships among protists.     

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional ¹³C‐edited ¹H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.     

Ultrastructural studies of the commensal suctorian,Choanophrya infundibulifera hartog

Summary The feeding tentacles of Choanophrya contain a central canal lined by microtubules. Only one tentacle develops during metamorphosis of the embryo into the adult, but others develop at intervals throughout adult life. Each tentacle forms adjacent to a solitary, subcortical kinetosome which lies parallel to the body surface, lacks accessory elements and never develops a cilium. Small condensations of electron-dense material and short bundles of microtubules form adjacent to the cartwheel region of the kinetosome. Initially these bundles are orientated randomly but later they become radially arranged and curved into prolamellae around a disc-shaped condensation centre, to form a paddlewheel-like tentacle primordium 0.8–1.1 m in diameter. The condensation centre consists of alternating concentric electron-dense and electron-transparent zones, and lies with its axis perpendicular to both the kinetosome and the cortex. The microtubules in each prolamella increase in number and pairs of short tip microtubules develop between adjacent prolamellae. Subsequently the developing lamellae become enclosed by a cylinder of ring microtubules. Once all the microtubule components of the tentacle primordium are established it increases in length by addition of material to the basal ends of the microtubules to form a short microtubule canal. As the canal elongates the epiplasm above it disappears and the pellicle membranes become uplifted around the protruding tentacle. An epiplasmic collar differentiates around the growing tentacle whilst spheroid vesicles and solenocysts begin to accumulate in the surrounding cytoplasm.This investigation was supported by the J.S. Dunkerley Fellowship in Protozoology, awarded by the University of Manchester.     

CELL WALL CHEMISTRY AND FINE STRUCTURE IN LEPTOIDS OF DENDROLIGOTRICHUM (BRYOPHYTA): THE END WALL

Leptoids (sieve elements) of Dendroligotrichum are characterized by a highly oblique end wall which is composed of cellulose (birefringent; IKI-H₂SO₄-positive), polyuronides (toluidine blue-positive), pectins (hydroxylamine-positive) and natural aldehydes (silver hexamine and silver proteinate-positive). Cytochemically the end wall appears identical to the unevenly thickened lateral wall. Electron cytochemical localization of aldehydes with silver proteinate reveals two distinct wall layers in comparison to the 3-layered lateral wall. Plasmodesmata are present in the end wall with a frequency of 15-20 per μm². A characteristic feature of end wall plasmodesmata is an expanded median cavity which is 0.12-0.15 μm in diameter. Frequently an electron-dense substance, whose chemical nature and origin are unknown, occludes the plasmodesmata.     

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.     

COMPARATIVE LEAF STRUCTURE OF SEVERAL SPECIES OF HOMOSPOROUS LEPTOSPORANGIATE FERNS

The structure of the mature leaves of 13 species from 9 families of homosporous leptosporangiate ferns was examined by light and electron microscopy. In 11 species (Adiantum pedatum L., Athyrium angustum Roth., Cyathea dregei Sm., Lygodium palmatum Sw., Mohria caffrorum (L.) Desv., Oleandra distenta Kuntae, Pellaea calomelanos (Sw.) Link, Pityrogramma calomelanos (L.) Link var. austro-americana (Domn.) Farw., Trichomanes melanotrichum Schlechtend., Vittaria guineensis Desv., and Woodwardia orientalis Sw.) the lamina veins are collateral; in two (Phlebodium aureum and Platycerium bifurcatum), bicollateral as well as collateral veins are present. The vascular bundles in the midribs of C. dregei and those in the petioles and midribs of Phlebodium and Platycerium are concentric. All of the vascular bundles in the homosporous leptosporangiate ferns studied are delimited by a tightly arranged cylinder of endodermal cells with Casparian strips. Within the veins without parenchymatic xylem sheaths, some sieve elements commonly abut tracheary elements with hydrolyzed primary walls. The majority of vascular parenchyma cells contact both sieve elements and tracheary elements, although some parenchyma cells are associated with only one type of conducting cell. Transfer cells (parenchyma cells with wall ingrowths) occur in the veins of 6 species examined. Most of the vascular parenchyma cells, however, have no distinctive structural characteristics. The sieve elements of the homosporous leptosporangiate ferns are very similar structurally and each consists of a plasmalemma, a parietal, anastomosing network of smooth endoplasmic reticulum (ER), and variable numbers of refractive spherules, plastids and mitochondria. The sieve elements of L. palmatum also contain plasmalemma tubules. The parenchymatic cells of the leaf (mesophyll, endodermal and vascular parenchyma cells) are united by desmotubule-containing plasmodesmata. The sieve elements are connected to each other by sieve pores and to parenchymatic cells by pore-plasmodesma connections. The sieve-area pores contain variable amounts of membranous material, apparently ER membranes, but do not occlude them. These membranes commonly are found in continuity with the parietal ER of the lumen. Based upon the relative frequencies of cytoplasmic connections between cell types, the photosynthates may move from the mesophyll to the site of phloem loading via somewhat different pathways in different species of homosporous leptosporangiate ferns.     

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.     

OBSERVATIONS ON PENETRATION OF LINDEN BRANCHES BY STYLETS OF THE APHID LONGISTIGMA CARYAE

Penetration of the bark of Tilia americana L., the linden tree, by Longistigma caryae (Harr.) is mainly intracellular. Like other aphids, L. caryae secretes a saliva sheath which encloses the path of the stylets, beginning with an external collar of sheath material on the surface of the periderm. Stylet sheaths within the bark gave positive reactions for callose, suggesting that, in reaction to wounding, punctured parenchyma cells secrete callose which diffuses throughout the stylet sheaths. Other, more conspicuous effects of wounding included: proliferation and enlargement of cells of the cortex and dilated rays bordering some stylet sheaths, formation of tylosoids in punctured sieve elements, deposition of massive amounts of callose in penetrated sieve elements and in sieve elements bordering penetrated cells, and stimulation of cambial activity and xylem differentiation. Stylet tips located in living sieve elements projected beyond their sheaths which terminated outside the sieve-element walls. It is suggested that such sieve elements can be considered to be functional. None of the living sieve elements containing stylet tips showed any signs of injury which could be attributed to the presence of the stylets. Stylet tips of feeding aphids were found in living sieve elements of both 1965 and 1966 phloem increments clearly indicating that L. caryae can feed on linden sieve elements more than 1 year of age.