SOME ULTRASTRUCTURAL ASPECTS OF METAPHLOEM SIEVE ELEMENTS IN THE AERIAL STEM OF THE HOLOPARASITIC ANGIOSPERM EPIFAGUS VIRGINIANA (OROBANCHACEAE)

A light and electron microscope investigation was conducted on phloem in the aerial stem of Epifagus virginiana (L.) Bart. Tissue was processed at field collection sites in an effort to overcome problems resulting from manipulation. At variance with earlier accounts, Epifagus phloem consists of sieve elements, companion cells, phloem parenchyma cells, and primary phloem fibers. The sieve elements possess simple sieve plates and the phloem is arranged in a collateral type of vascular bundle. In addition, this constitutes the first study on phloem ultrastructure in the aerial stems of a holoparasitic dicotyledon, an entire plant which could be viewed as an “ideal sink.” Epifagus phloem possesses unoccluded sieve plate pores in mature sieve elements and a total lack of P-protein in sieve elements at all stages of development. Mature sieve elements lack nuclei. Plastids were rarely observed in mature sieve elements. Vacuoles with intact tonoplasts were encountered in some mature sieve elements. Otherwise, the ultrastructural features of sieve elements appear to differ little from those described by investigators of non-parasitic species.     

OBSERVATIONS ON SIEVE ELEMENTS IN THREE PERENNIAL MONOCOTYLEDONS

Seasonal collections were made of rhizomes of Polygonatum canaliculatum and Typha latifolia and of aerial stems of Smilax hispida. Many metaphloem sieve elements in all three species remain functional for 2 or more years, or for the life of the plant parts in which they occur. Although the protoplasts of mature sieve elements remain similar in appearance from one time of year to the next, the amount of callose associated with the sieve plates and lateral sieve areas of such cells apparently varies with the seasons, being heavier in late fall and winter and lighter in late spring and summer. At maturity the metaphloem sieve elements contain strands derived from the slime bodies of immature cells. It is suggested that in mature sieve elements the slime strands normally occur as a network along the wall. Many mature sieve elements of S. hispida contained normal-appearing nuclei.     

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.     

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.     

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.     

Sieve element characters and the systematic position ofAustrobaileya,Austrobaileyaceae—with comments to the distinction and definition of sieve cells and sieve-tube members

The ultrastructure of the sieve elements ofAustrobaileya is compared with that of angiosperm sieve tubes and gymnosperm sieve cells (mostly fromCycadales). Except for the size of the sieve poresAustrobaileya shares all ultrastructural characters (e.g., chromatolytic nuclear degeneration, presence of p-protein, formation of sieve pores from unbranched plasmodesmata) and other features (e.g., companion cells) with angiosperm sieve tubes. Gymnosperm sieve cells on the contrary are characterized by pycnotic nuclear degeneration, absence of p-protein, formation of sieve areas from branched plasmodesmata with median cavities. — The exact ordinal assignment ofAustrobaileya within the subclassMagnoliidae is still disputed, a placement close to eitherMyristicaceae andWinteraceae orMonimiaceae being possible as judged from both S-type sieve-element plastids and p-protein bodies. — On the basis of the ultrastructural results fromAustrobaileya it is proposed to reconsider concepts and terminology of sieve elements. i.e., to include features from sieve pore development, nuclei degeneration and presence of specific proteins into the definitions and to restrict the term sieve cell to gymnospermous sieve elements which differ much from those of other vascular plants.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

THE FINE STRUCTURE OF SIEVE ELEMENTS OF NEREOCYSTIS LÜTKEANA

The sieve elements of Nereocystis from the base of phylloids contain numerous small vesicles, cytoplasm, ribosomes, and the usual organelles and membrane systems, including nuclei, plastids, mitochondria, dictyosomes, and endoplasmic reticulum. They have a thick secondary wall layer which is deposited along the longitudinal walls and at the sieve plate excluding the sieve pores. The sieve pores range in diameter from 100 to 400 nm and are lined by plasmalemma. The sieve elements from the hollow basal parts of the pneumatocyst show essentially the same features but have larger and fewer vesicles, relatively little cytoplasm, larger sieve pores, 400–900 nm in diameter, and may lack a nucleus. In old sieve elements there are large deposits of callose on the sieve plate and along the longitudinal wall; the vesicles seem to break down, and the protoplast appears necrotic. It is concluded that the trumpet hyphae and sieve tubes are basically the same type of cell, and that the trumpet-shape of the sieve elements is due to their passive stretching during extension growth of the organ in which they occur. There are minor but significant differences among the sieve elements from different regions of the thallus which may reflect possible levels of structural specialization of the sieve elements within the same plant.     

Sieve elements rapidly develop ‘nacreous walls’ following injury − a common wounding response?

Thick glistening cell walls occur in sieve tubes of all major land plant taxa. Historically, these ‘nacreous walls’ have been considered a diagnostic feature of sieve elements; they represent a conundrum, though, in the context of the widely accepted pressure–flow theory as they severely constrict sieve tubes. We employed the cucurbit Gerrardanthus macrorhizus as a model to study nacreous walls in sieve elements by standard and in situ confocal microscopy and electron microscopy, focusing on changes in functional sieve tubes that occur when prepared for microscopic observation. Over 90% of sieve elements in tissue sections processed for microscopy by standard methods exhibit nacreous walls. Sieve elements in whole, live plants that were actively transporting as shown by phloem‐mobile tracers, lacked nacreous walls and exhibited open lumina of circular cross‐sections instead, an appropriate structure for Münch‐type mass flow of the cell contents. Puncturing of transporting sieve elements with micropipettes triggered the rapid (<1 min) development of nacreous walls that occluded the cell lumen almost completely. We conclude that nacreous walls are preparation artefacts rather than structural features of transporting sieve elements. Nacreous walls in land plants resemble the reversibly swellable walls found in various algae, suggesting that they may function in turgor buffering, the amelioration of osmotic stress, wounding‐induced sieve tube occlusion, and possibly local defence responses of the phloem.     

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

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

Polysomes and intracisternal accumulations in enucleate sieve elements of rice (Oryza sativa L.)

Polysomes in sieve elements of rice (Oryza sativa L.) were studied with the electron microscope. The polysomes were found on the rough endoplasmic reticulum (ER) present in immature sieve elements and also on the cisternae of aggregated ER in the parietal layer of mature, enucleate sieve elements. In the immature sieve elements the ER cisternae existed as narrow profiles while in the mature sieve elements the ER cisternae were considerably dilated and contained a fibrillar material and, occasionally, electron-opaque inclusions. In addition to the aggregated ER, single profiles of ER were found applied to the lateral walls and also the sieve plates. These cisternae also bore ribosomes and were separated from the plasmalemma by a narrow, dense space. In the mature sieve elements much of the surface of the ER membranes was covered with polysomes. The dimensions of the polysomes are described and the possibility that they contribute to the formation of the fibrillar material in the intracisternal space is discussed.Abbreviations ER endoplasmic reticulum     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

CALLOSE SUBSTANCE IN SIEVE ELEMENTS

The secondary phloem of 6 species of woody dicotyledons was examined for the occurrence of callose on the sieve plates of active sieve elements. Fluorescence and bright-field staining methods were used to detect callose. Tissue from the 6 species was killed and fixed in each of 5 solutions. Some tissue of each species was submerged in the killing solutions as quickly as possible, the remainder within 15 min after removal from the tree. In each species, some active sieve elements of the quick-killed tissue gave negative callose reactions. All active sieve elements of the delay-killed tissue gave positive callose reactions. These and other results suggest that the active sieve elements in the secondary phloem of the species studied normally lack callose and that the extent of callose deposition in these cells depended primarily upon the rapidity with which the sieve-element protoplasts were killed after wounding of the phloem. In addition, bright-field observations of sieve plates of large numbers of sieve elements from a seasonal collection of Tilia americana secondary phloem suggest that the active sieve elements normally lack callose during the growing season and that the inactive sieve elements normally possess it (dormancy callose).     

STRUCTURE AND DEVELOPMENT OF THE SIEVE ELEMENT IN THE STEM OF LYCOPODIUM LUCIDULUM

Stem tissue of Lycopodium lucidulum Michx. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. Although their protoplasts contain similar components, immature sieve elements can be distinguished from parenchymatous elements of the phloem at an early stage by their thick walls and correspondingly high population of dictyosomes and dictyosome vesicles. Late in maturation the sieve-element walls undergo a reduction in thickness, apparently due to an “erosion” or hydrolysis of wall material. At maturity, the plasmalemma-lined sieve elements contain plastids with a system of much convoluted inner membranes, mitochondria, and remnants of nuclei. Although the endoplasmic reticulum (ER) in most mature sieve elements was vesiculate, in the better preserved ones the ER formed a tubular network closely appressed to the plasmalemma. The sieve elements lack refractive spherules and P-protein. The protoplasts of contiguous sieve elements are connected with one another by pores of variable diameter, aggregated in sieve areas. As there is no consistent difference between pore size in end and lateral walls these elements are considered as sieve cells.     

Spatial and temporal regulation of the forisome gene <Emphasis Type="Italic">for1</Emphasis> in the phloem during plant development

Forisomes are protein aggregates found uniquely in the sieve elements of Fabaceaen plants. Upon wounding they undergo a reversible, calcium-dependent conformational switch which enables them to act as cellular stopcocks. Forisomes begin to form in young sieve elements at an early stage of metaphloem differentiation. Genes encoding forisome components could therefore be useful as markers of early sieve element development. Here we present a comprehensive analysis of the developmental expression profile of for1, which encodes such a forisome component. The for1 gene is highly conserved among Fabaceaen species and appears to be unique to this phylogenetic lineage since no orthologous genes have been found in other plants, including Arabidopsis and rice. Even so, transgenic tobacco plants expressing reporter genes under the control of the for1 promoter display reporter activity exclusively in immature sieve elements. This suggests that the regulation of sieve element development is highly conserved even in plants where mature forisomes have not been detected. The promoter system could therefore provide a powerful tool for the detailed analysis of differentiation in metaphloem sieve elements in an unexpectedly broad range of plant species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Plastids in sieve elements and companion cells of Tilia americana

Summary Contrary to an earlier report, the sieve elements and companion cells of Tilia americana contain plastids. In young sieve elements and companion cells the plastids contain a moderately electronopaque matrix and internal membranes; the latter are very numerous in the plastids of the sieve elements. Plastids of mature sieve elements contain an electron-transparent matrix, apparently fewer internal membranes than the plastids of young elements, and a single starch grain each. The plastids of companion cells undergo little or no structural modification during cellular differentiation, and apparently contain no starch.This research has been supported by the National Science Foundation, grants GB-5950 and GB-8330.     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

ULTRASTRUCTURE OF THE NACREOUS LEPTOIDS (SIEVE ELEMENTS) IN THE POLYTRICHACEOUS MOSS ATRICHUM UNDULATUM

The physiological phloem equivalents, leptoids, of the polytrichaceous moss Atrichum undulatum appear to be similar to the nacreous sieve elements that occur in many higher plants. These leptoids are elongated cells with nacreous thickenings on their radial and tangential walls. Their oblique end walls, which lack such thickenings, are traversed by numerous pores through which the plasmalemma, endoplasmic reticulum, and cytoplasm are continuous between adjacent leptoids of a longitudinal file. These end walls closely resemble the simple sieve areas of the sieve elements found in Polypodium vulgare. The leptoid sieve pores have a median expanded area and frequently are occluded by small amorphous protein plugs at each end. Also, callose was observed as electron-luscent areas both on the faces of the end walls and as a thin cylinder surrounding the lateral area of each pore. Amorphous and granular cytoplasmic contents of the leptoids appear to be morphologically similar to the slime (P-protein) found in the sieve-tube elements of many angiosperms. Differentiating leptoids are characterized by the formation of numerous membrane-bound protein bodies in close association with polysomes and endoplasmic reticulum. As the leptoid matures, the contents of the protein bodies become dispersed in the cytoplasm. Ultrastructurally and ontogenetically the leptoids in the gametophores of A. undulatum appear almost identical to the sieve elements of P. vulgare and therefore should be considered sieve elements rather than phloem-like equivalents.     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.