ONTOGENY AND STRUCTURE OF THE SECONDARY PHLOEM IN EPHEDRA

The secondary phloem in Ephedra is atypical of the gymnosperms in general and exhibits several angiosperm-like characteristics. The ray system of the conducting phloem consists of parenchymatous, multiseriate rays. The axial system contains parenchyma cells, sieve cells, and unusual albuminous cells reminiscent of the specialized parenchyma cells found in some angiosperms. These cell types may intergrade with each other. P-protein in the developing sieve element appears early in the form of a single, ovoid slime body. Later, smaller slime bodies appear and quickly disperse. In the mature sieve element the single, ovoid slime body is lost, and P-protein is then evident in the form of a parietal cylinder, thread-like strands, amorphose globules, or a slime plug. Necrotic-appearing nuclei are commonly found in mature sieve cells.     

Development and Structure of Phloem in the Petiole of Lagenaria siceraria (Mol.) Standl. and Momordica charantia L.

Light microscopic studies of the petioles of Lagenaria sicerariareveal that the external phloem of each bicollateral vascularbundle develops earlier than the internal phloem, and that thesieve elements of the external phloem are arranged in the outerand inner zones. Each sieve element of L. siceraria and Momordicacharantia is vertically associated with a maximum of six andtwo companion cells respectively. Discrete granular bodies seenin the cytoplasm of young sieve elements develop into globular,oval, or elongated slime bodies. Enlargement and fusion of slimebodies, and the subsequent dispersal of slime occur in the parietalcytoplasm. The dispersal of slime coincides with degradationof the nucleus and perforation of the pore sites. Before nucleardisorganization, the sieve-element nucleolus is extruded. Slimeafter its immediate dispersal appears amorphous and uniformlydistributed in the sieve elements. Plugs exhibit varying degreesof condensation of slime near the sieve plates. Certain maturesieve elements in the external phloem of L. siceraria have ovalbodies which we consider reaggregated or undispersed slime.Evidence has been obtained that a central cavity occurs in afew, almost mature, sieve elements wherein the cytoplasm includingthe slime is peripheral.     

Another View of the Ultrastructure of Cucurbita Phloem

The primary phloem of young internodes of Cucurbita maxima wasstudied with the electron microscope. Phloem parenchyma cellsare highly vacuolated and contain nuclei, endoplasmic reticulum,ribosomes, mitochondria, chloro-plasts, and occasional dictyosomes.As compared with parenchyma cells, the most distinctive featuresof companion cells are their extremely dense cytoplasm, lowdegree of vacuolation, lack of chloroplasts, and numerous sieve-elementconnexions. Companion cells contain plastids with few internalmembranes. At maturity the enucleate sieve element is linedby a plasmalemma, one or more cistema-like layers of endoplasmicreticulum, and a membrane which apparently delimits the parietallayer of cytoplasm from a large central cavity. In OsO_4–-andglutaraldehyde-fixed elements, the central cavity is traversedby numerous strands, which run from cell to cell through thepores of sieve plates and lateral sieve areas, and which arederived ontogenetically from the slime bodies of immature cells.Numerous normal-appearing mitochondria are present in the parietallayer of cytoplasm. The pores of sieve plates and lateral sieveareas are lined with cytoplasm. The ultrastructural detailsof young sieve elements differ little from those of other youngnucleate cells. During sieve-element development, the sieveelement increases in vacuolation. At the same time, slime bodiesdevelop in the cytoplasm. With glutaraldehyde fixation, thesebodies often exhibit a double-layered limiting membrane. Asthe sieve element continues to differentiate, the slime bodiesincrease in size and the parietal layer of cytoplasm becomesvery narrow. Presently, the slime bodies begin to disperse andtheir contents fuse. This phenomenon occurs in the parietallayer of cytoplasm, while the latter is still delimited fromthe large central vacuole by a distinct tonoplast. The initiationof slime-body dispersal more or less coincides with perforationof the pore sites, and many pores are traversed by slime earlyin their development. Before slime-body dispersal, all dictyosomesand associated vesicles disappear from the cytoplasm. Eventually,the tonoplast diappears and the slime becomes distributed throughoutthe central cavity in the form of strands. Nuclei and ribosomesdisappear before breakdown of the tonoplast. Sieve elementsare connected with companion cells and parenchyma cells by plasmodesmata.     

SOME ASPECTS OF SIEVE CELL ULTRASTRUCTURE IN PINUS STROBUS

The immature sieve cell of Pinus strobus contains all of the protoplasmic components commonly encountered in young cell types. In addition, it contains slime bodies with distinct double-layered limiting membranes. The mature sieve cell is lined by a narrow layer of cytoplasm consisting of a plasmalemma, one or more layers of anastomosing tubules of endoplasmic reticulum, numerous mitochondria, starch granules and crystal-like bodies. Each mature cell contains a necrotic nucleus. Ribosomes and dictyosomes are lacking. Strands derived ontogenetically from the slime bodies of the immature cell traverse the central cavity and are continuous with those of neighboring sieve cells through the plasmalemma-lined pores of the sieve areas. Sieve-area pores are also traversed by numerous endoplasmic membranes. A membrane was not found separating the parietal layer of cytoplasm from the large central cavity.     

ULTRASTRUCTURE OF THE SECONDARY PHLOEM OF TILIA AMERICANA

Summer and winter (July and January) samples of secondary phloem of Tilia americana were studied with the electron microscope. Parenchyma cells contain: nuclei, endoplasmic reticulum, ribosomes, plastids, mitochondria and occasional dictyosomes. Well-defined tonoplasts separate vacuoles from cytoplasmic ground substance. Vacuoles often contain tannins. Lipid droplets are common in cytoplasm. Endoplasmic reticulum–connected plasmodesmata are aggregated in primary pit fields. Companion cells differ from parenchyma cells in having numerous sieve-element connections, possibly slime, and in lacking plastids. Mature, enucleate sieve elements possess 1–4 extruded nucleoli. Numerous vesicles occupy a mostly parietal position in association with plasmalemma. The mature sieve element lacks endoplasmic reticulum, organelles (except for few mitochondria) and tonoplast. In OsO_4– and glutaraldehyde-fixed elements, slime has a fine, fibrillar appearance. Normally, these fine fibrils are organized into coarser ones which form strands that traverse the cell and the plasmalemma-lined pores of sieve plates and lateral sieve areas.     

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.     

Observations on Companion Cells and Specialized Phloem Parenchyma Cells of Nelumbo nucifera Gaertn

The phloem of very young petioles of Nelumbo nucifera Gaertn.(Nelumbium speciosum Willd.) was studied with the light microscope.The elongated, mature sieve elements contain slime, plugs, strands,and numerous plastids. Some sieve elements remain nucleatedfor a brief period even after the sieve plates are well developed.The companion cells numbering 8–14 undergo disintegrationbefore the elongation of the ontogenetically related sieve elementis completed. They are uninucleate to begin with but later becomebinucleate and finally degenerate and obliterate. The variousstages in their ontogeny and disintegration are described. Ofthe very few specialized phloem parenchyma cells present, someare associated with sieve elements. They have slime body-likestructures, and plastid-like bodies which group together andeventually disintegrate.     

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.     

Penetration of faba bean sieve elements by pea aphid does not trigger forisome dispersal

Immediately after their stylets penetrate a phloem sieve element, aphids inject saliva into the sieve element for approximately 30–60 s before they begin to ingest phloem sap. This salivation period is recorded as waveform E1 in electrical penetration graph (EPG) monitoring of aphid feeding behavior. It has been hypothesized that the function of this initial period of phloem salivation is to reverse or prevent plugging of the sieve element by one of the plant's phloem defenses: formation of P‐protein plugs or callose synthesis in the sieve pores that connect adjacent sieve elements. This hypothesis was tested using the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), and faba bean, Vicia faba L. (Fabaceae), as a model system, and the results do not support the hypothesis. In legumes, such as faba bean, P‐protein plugs in sieve elements are formed by dispersal of proteinaceous bodies called forisomes. Contrary to the hypothesis, the great majority of sieve element penetrations by pea aphid stylets do not trigger forisome dispersal. Thirteen sieve elements were cryofixed early in phloem phase before the aphids could complete their salivation period and the forisomes were not dispersed in any of the 13 samples. However, in these samples, the aphids completed on average a little over half of their normal E1 salivation period before they were cryofixed. Thus, it is possible that sieve element penetration triggered forisome dispersal in these samples but the abbreviated period of salivation was still sufficient to reverse dispersal. To rule out this possibility, 17 sieve elements were cryofixed during R‐pds, which are an EPG waveform associated with sieve element penetration but without the characteristic E1 salivation that occurs during phloem phase. In 16 of the 17 samples, the forisomes were not dispersed. Thus, faba bean sieve elements usually do not form P‐protein plugs in response to penetration by pea aphid stylets. Consequently, the characteristic E1 salivation that occurs at the start of each phloem phase does not seem to be necessary to prevent a plugging response because penetration of sieve elements during R‐pds does not trigger forisome dispersal despite the absence of E1 salivation. Furthermore, as P‐protein plugs do not normally form in response to sieve element penetration, E1 salivation that occurs at the start of each phloem phase is not a response to development of a P‐protein plug. Thus, the E1 salivation period at the beginning of the phloem phase appears to have function(s) unrelated to phloem sealing.     

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     

STRUCTURE AND DEVELOPMENT OF SIEVE ELEMENTS IN THE LEAF OF ISOETES MURICATA

Leaf tissue of Isoetes muricata Dur. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. The very young sieve elements can be distinguished from contiguous parenchyma cells by their distinctive plastids and the presence of crystalline and fibrillar proteinaceous material in dilated cisternae of the rough ER. During differentiation, the portions of ER enclosing this proteinaceous substance become smooth surfaced and migrate to the cell wall. Along the way they apparently form multivesicular bodies which then fuse with the plasmalemma, discharging their contents to the outside. At maturity, the sieve element contains an elongate nucleus, which consists of dense chromatin material, and remnants of the nuclear envelope. In addition, the mature sieve element is lined by a plasmalemma and a parietal, anastomosing network of smooth ER. Both plastids and mitochondria are present. P-protein is lacking at all stages of development. Tonoplasts are. not discernible in mature sieve elements. The end walls of mature sieve elements contain either plasmodesmata or sieve pores or both, but only plasmodesmata occur in the lateral walls.     

Observations on albuminous cells in Pinus

Summary Albuminous cells were examined in seasonal collections of secondary phloem of Pinus banksiana, P. resinosa and P. strobus. It was determined that the only reliable criterion for the identification of albuminous cells is their conspicuous connections with sieve cells. Discrete bodies, which give positive protein reactions, were encountered in young albuminous cells. These bodies, which have been interpreted as slime bodies, eventually elongate in the cytoplasm and disappear. During periods of high starch content in other parenchymatous elements, albuminous cells may also contain starch.This research has been supported by National Science Foundation grant GB-3193.     

ANATOMICAL FEATURES OF METAPHLOEM IN STEMS OF SABAL,COCOS AND TWO OTHER PALMS

Sieve tubes in metaphloem of palm stems function throughout the life of the plant and merit close investigation. A stem of Sabal palmetto estimated to be 50 years old was sampled extensively. Variation in length of sieve-tube elements throughout this stem was measured and is discussed. In the metaphloem of individual vascular bundles companion cells are not sharply differentiated from other phloem parenchyma cells. Definitive callose deposits and slime are normally absent from mature sieve tubes, even in fixed material. Otherwise no conspicuous structural features which might account for the longevity of sieve tubes can be discerned. Occlusion of phloem strands after leaf fall is initially by callose deposition on sieve plates followed immediately by tylosoid formation. Similar sampling of Cocos nucifera, Washingtonia robusta and to a lesser extent Archontophoenix alexandrae confirmed these results except for quantitative differences.     

ONTOGENY AND STRUCTURE OF THE SECONDARY PHLOEM IN PYRUS MALUS

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

SEASONAL DEVELOPMENT OF THE SECONDARY PHLOEM IN PINUS

Functional sieve cells are present at all times in the secondary phloem of Pinus banksiana Lamb., P. resinosa Ait., and P. strobus L. With regard to a given year's growth increment, all but the last-formed sieve cells (2-4 layers) cease functioning the same season they are derived from the cambium. The former overwinter and remain functional until new sieve cells differentiate in spring. Toward the end of March undifferentiated cells in the outer margin of the cambial zone begin to differentiate into sieve cells. About a week later, cambial activity (cell division) commences. All early phloem is produced by early May before new xylem differentiation begins. Most sieve cells are differentiated by late August, but a few not until late September. Cessation of function begins in late May or June with formation of definitive callose on sieve areas of the sieve cells which overwintered and continues slowly to sieve cells of the current season's early phloem. By mid-December all but the last-formed sieve cells (i.e., those which will overwinter in a functional state) are devoid of contents. Phloem differentiation precedes xylem differentiation by approximately 1 1/2 months. Xylem and phloem production cease more or less simultaneously in August, xylem and phloem differentiation in September.     

THE SEASONAL CYCLE OF PHLOEM DEVELOPMENT IN JUNIPERUS CALIFORNICA

In Juniperus californica, all sieve cells of the previous season's phloem growth increment overwinter in a mature state. Initiation of cambial activity begins in early March and, by the end of March, the oldest sieve cells that overwintered lose their contents and die. By mid-April, even the youngest sieve cells of the previous season's growth increment have lost their contents. The period of greatest cambial activity begins in the last half of April and continues through May. With the slowing of cambial activity in June, callose begins to collect on the sieve areas of the first-formed sieve cells of the new increment. By July, the cambium and phloem are in a dormant state. Initiation of phloem production precedes that of the xylem by about 1 month. Production of new xylem and phloem ceases simultaneously in July.     

PHLOEM OF SPHENOPHYLLUM

The phloem of most fossil plants, including that of Sphenophyllum, is very poorly known. Sphenophyllum was a relatively small type of fossil arthrophyte with jointed stems bearing whorls of leaves ranging in form from wedge or fan-shaped to bifid, to linear. The aerial stem systems of the plant exhibited determinate growth involving progressive reduction in the dimensions of the stem primary bodies, fewer leaves per whorl, and smaller and simpler leaves distally. The primary phloem occurs in three areas alternating in position with the arms of the triarch centrally placed primary xylem. Cells of the primary phloem, presumably sieve elements, are axially elongate with horizontal to slightly tapered end walls. In larger stems with abundant secondary xylem and secondary cortex or periderm, a zone of secondary phloem occurs whose structure varies in the three areas opposite the arms of the primary xylem, as opposed to the three areas lying opposite the concave sides of the primary xylem. The axial system of the secondary phloem consists of vertical series of sieve elements with horizontal end walls. In the areas opposite the protoxylem the parenchyma is present as a prominent ray system showing dilation peripherally. Sieve elements in the areas opposite the protoxylem arms have relatively small diameters. In the areas between the protoxylem poles the secondary phloem sieve elements have large diameters and are less obviously in radial files, while the parenchyma resembles that of the secondary xylem in these areas in that it consists of strands of cells extending both radially and tangentially. An actively meristematic vascular cambium has not been found, indicating that this layer changed histologically after the cessation of growth in the determinate aerial stem systems and was replaced by a post-meristematic parenchyma sheath made up of axially elongate parenchyma lacking cells indicative of being either fusiform or ray initials. A phellogen arose early in development in a tissue believed to represent pericycle and produced tissue comparable to phellem externally. Normally, derivatives of the phellogen underwent one division prior to the maturation of the cells. Concentric bands of cells with dark contents apparently represent secretory tissue in the periderm and cell arrangements indicate that a single persistent phellogen was present. Sphenophyllum is compared with other arthrophytes as to phloem structure and is at present the best documented example of a plant with a functionally bifacial vascular cambium in any exclusively non-seed group of vascular plants.     

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.     

ONTOGENY AND STRUCTURE OF CONIFEROUS SIEVE CELLS

Studies of the secondary phloem of 6 species of conifers revealed that mature sieve-cell protoplasts contain internal strands which are derived ontogenetically from slime bodies of immature cells. These strands, each measuring about 0.3 μ in diameter, traverse the cell and run from cell to cell through sieve-area pores. Coniferous sieve cells have much in common—both ontogenetically and structurally—with dicotyledonous sieve-tube members.     

OBSERVATIONS ON METAPHLOEM IN THE VEGETATIVE PARTS OF PALMS

Metaphloem was studied in available vegetative parts of 374 species in 164 genera of palms. Sieve elements usually have compound sieve plates except in the subfamilies Lepidocaryoideae and Nypoideae. Sieve elements in roots usually have oblique to very oblique end walls, whereas in stems and leaves they have transverse to oblique walls. Within a phloem strand the degree of compounding of a sieve plate is directly correlated with element diameter. Plastids are normally present in functioning, enucleate sieve elements. Small quantities of “slime” substances have been detected in young sieve elements in stems and petioles of a few species. Many sieve plates in functioning sieve elements lacked callose in materials quick-killed in liquid nitrogen or chilled acetic-alcohol. Definitive callose is confined to sieve elements just before their obliteration. Sieve tubes in leaf and stem are usually ensheathed by contiguous parenchyma cells while those in root have very few contiguous parenchyma cells. Two types of contiguous parenchyma cells can be distinguished by difference in cytoplasmic density, especially with the electron microscope. Cells with denser cytoplasm are interpreted as companion cells. Lignified contiguous parenchyma cells are occasionally present in metaphloem of petioles. The possible diagnostic and taxonomic features of metaphloem are discussed.