SIEVE-ELEMENT ONTOGENY IN THE ROOT OF EQUISETUM HYEMALE

Roots of Equisetum hyemale L. var. affine (Engelm.) A. A. Eat. were fixed in glutaraldehyde, postfixed in osmium tetroxide, and sieve elements of various ages were examined with the electron microscope. Young sieve elements are distinguished by their position within the vascular cylinder and by the presence of numerous refractive spherules, which originate within dilated portions of the endoplasmic reticulum (ER). Early in development, the sieve-element walls undergo a substantial increase in thickness. This is followed by the appearance of massive ER aggregates in the cytoplasm and then by a phase involving stacking and sequestering of the remaining ER. Nuclear degeneration is initiated shortly after the appearance of the ER aggregates. The chromatin condenses into masses of variable size along the inner surface of the nuclear envelope. The envelope then ruptures and chromatin is released into the cytoplasm. During the period of nuclear degeneration, mitochondria and plastids undergo structural modification, while components such as dictyosomes, microtubules, and ribosomes degenerate and disappear. The remaining cytoplasmic components assume a parietal position in the cell, leaving the lumen of the cell clear in appearance. At maturity, the plasmalemma-lined sieve element contains plastids, mitochondria, some ER, and refractive spherules. At this time many of the refractive spherules are discharged into the region of the wall. Pores between sieve elements occur largely on the end walls. During pore development, tubules of ER apparently traverse the pores, but because of the presence of massive callose deposits in the material examined, the true condition of mature pores could not be determined. The connections between mature sieve elements and pericycle cells are characterized by the presence of massive wall thickenings on the pericycle-cell side. Plasmodesmata in the wall thickening are matched by pores on the sieve-element side. Ontogenetic and cytoplasmic factors argue against use of the term “companion cell” for the vascular parenchyma cells associated with the sieve elements.     

Sieve-Element Ontogeny in the Aerial Shoot of Equisetum hyemale L.

The aerial shoots of Equisetum hyemale L. var. affine (Engelm.)A. A. Eat. were examined with the electron microscope as partof a continuing study of sieveelement development in the lowervascular plants. Young E. hyemale sieve elements are distinguishablefrom all other cell types within the vascular system by thepresence of refractive spherules, proteinaceous bodies whichdevelop within dilated portions of the endoplasmic reticulum(ER). Details of cell wall thickening differ between protophloemand metaphloem sieve elements. Following cell wall thickeningthe ER increases in quantity and aggregates into stacks. Shortlythereafter, nuclear degeneration is initiated. During the periodof nuclear degeneration some cytoplasmic components-dictyosomes,microtubules and ribosomes-degenerate and disappear, while organellessuch as mitochondria and plastids persist. The latter undergostructural modifications and become parietal in distribution.Eventually the massive quantities of ER are reduced, leavingthe lumen of the cell clear in appearance. At maturity the plasmalemma-linedsieve element contains a parietal network of tubular ER, aswell as mitochondria, plastids, and refractive sphemh At thistime many of the spherules are discharged into the region ofthe wall. Sieveelement pores occur in both lateral and end walls.At maturity many pores are traversed by large numbers of ERmembranes. The metaphloem sieve elements of the mid-internodalregions apparently are sieve-tube members. The connections betweenmature protophloem sieve elements and pericycle cells are associatedwith massive wall thickenings on the pericyclecell side.     

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.     

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.     

STRUCTURE AND DEVELOPMENT OF THE SIEVE ELEMENTS IN PSILOTUM NUDUM

Shoot tissue of Psilotum nudum (L.) Griseb. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. Young sieve elements can be distinguished from contiguous parenchyma cells by their distinctive plastids, the presence of refractive spherules, and the overall dense appearance of their protoplast. The refractive spherules apparently originate in the intracisternal spaces of the endoplasmic reticulum (ER). With increasing age the sieve-element wall undergoes a marked increase in thickness. Concomitantly, a marked increase occurs in the production of dictyosome vesicles, many of which can be seen in varying degrees of fusion with the plasmalemma. Other fibril- and vesicle-containing vacuoles also are found in the cytoplasm. In many instances the delimiting membrane of these vacuoles was continuous with the plasmalemma. Vesicles and fibrillar materials similar to those of the vacuoles were found in the younger portions of the wall. At maturity the plasmalemma-lined sieve element contains a parietal network of ER, plastids, mitochondria, and remnants of nuclei. The protoplasts of contiguous sieve elements are connected by solitary pores on lateral walls and pores aggregated into sieve areas on end walls. All pores are lined by the plasmalemma and filled with numerous ER membranes which arise selectively at developing pore sites, independently of the ER elsewhere in the cell. P-protein and callose are lacking at all stages of development.     

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.     

Le phloème deSelaginella willdenowii: histophysiologie comparée

Metaphloem sieve elements ofSelaginella willdenowii are elongated cells with slightly oblique or transverse end walls. Pores are seen on both lateral and end walls, although they are more numerous on the latter. Parenchyma cells exhibiting strong enzyme activities (acid phosphatase, non specific esterase, succinate dehydrogenase, cytochrome oxidase, peroxidase) are present between sieve elements and tracheids in each vascular bundle. A functional association thus appears to exist between these parenchyma cells and the conducting elements.—The occurrence of transverse to slightly oblique end walls in sieve elements seems to characterize the ligulate Lycopsids (as opposed to the aligulateLycopodium where sieve elements possess slanting, very oblique, end walls).

     

Some aspects of sieve-element structure and development inSelaginella kraussiana

Summary The vacuoles of the sieve elements ofSelaginella kraussiana contain a crystalline protein which appears to degenerate in mature cells. Although it occurs in sieve elements we have elected not to call it P-protein because of ontogenetic and possibly functional differences between the two. The nucleus undergoes unique structural changes during development of the sieve element, ultimately being converted to a mass of tubules. The structures referred to by earlier workers as refractive spherules inSelaginella are probably plastids. As the size of the sieve pores in lateral and end walls falls into the same size range, the sieve elements ofSelaginella kraussiana can be considered to be sieve cells.This work was supported by the U.S. National Science Foundation (GB 31417).     

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.     

Form-Pfs Plastids,Stem Anatomy and Systematic Affinities of Stylobasium Desf. (Stylobasiaceae)

The vascular system of the stem of Stylobasium was investigated during its primary and secondary phases with both light and electron microscopic methods. It contains collateral bundles arranged in a ring, separated by rays which undergo regular cambial growth. The phloem consists of short sieve elements connected to sieve tubes by simple sieve plates, companion cells of the same length, and phloem parenchyma cells. During their autophagy-like differentiation and maturation, typical of all angiosperms, the sieve elements of Stylobasium have a peculiar feature, whereby they develop and retain form-Pfs plastids (containing protein filaments and starch). The sieve-element plastids of the two Stylobasium species, and of some 100 species belonging to taxa of which Stylobasium had been considered to be a possible member, have been studied by transmission electron microscopy. With the exception of a few species with form-Pcs plastids (containing a single small protein crystal in addition to starch), the great majority of taxa studied are characterized by S-type sieve-element plastids (containing starch only). The presence of form-Pfs plastids in Stylobasium supports its separation into the unigeneric Stylobasiaceae and the placement of this family close to other form-Pfs or form-Pcfs-containing taxa. While other characters would exclude an affiliation to the Magnolianae (form-Pfs plastids in Canella) or Caryophyllales (form-Pfs plastids in Microtea), an association with the form-Pcfs families Connaraceae and Mimosaceae is positively considered and corresponds to their frequent allocation close to the Rutales and Sapindales. Within the Rutales/Sapindales the sizes of sieve-element plastids (average diameter) range from very large (e.g. in the Julianaceae) to comparatively small (e.g. in Aceraceae) and are used to group the families. The sieve element characters of the Coriariaceae (tiny plastids with almost no starch, wide sieve plate pores, copious P-protein) suggest their removal from Rutales/Sapindales into the neighbourhood of the Cucurbitaceae.     

Ricerche Sull'infrastruttura del Coleottile di Avena

Abstract

Researches on ultrastructure of Avena coleoptile. 3. The sieve elements. — A study on the ultrastructural organization of the mature sieve elements of Avena coleoptile has been carried out. Data suggest that functional phloem tubes are alive and remain alive until they are working. Judging on morphological basis, the metabolic activity of sieve elements should be of peculiar type and low in comparison to that of the companion cells. In fact the cytoplasm is located in a narrow parietal strand, mitochondria, Golgi apparatus and endoplasmic reticulum are present, but they appear very modified; plastids and nucleus are absent. The cytoplasm is bounded externally by a normal plasmalemma, whilst the vacuole has no visible limits: a tonoplast is, therefore not identifiable.

The strands connecting the superimposed sieve elements with one another through the sieve plate result to be made by a double membrane system very similar to the endoplasmic reticulum, which we believe to realize cytoplasmic continuity between phloem tubes.

The data reported are more favorable to the existence in the sieve tubes of an active mechanism of translocation of organic solutes than a passive mass-flow.

The collaboration of companion cells in the translocation mechanism has been discussed.     

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.     

TRANSLOCATION IN MACROCYSTIS. III. COMPOSITION OF SIEVE TUBE EXUDATE AND IDENTIFICATION OF THE MAJOR C14-LABELED PRODUCTS1

The major C¹⁴-labeled substance in sieve tube exudate of M. pyrifera is D-mannitol, comprising 3.6% (w/v). No sugars are detectable. Certain amino acids also possess some C¹⁴-labeling and occur in significantly high concentrations in exudate. The exudate contains negligible ether-soluble lipid, but has a large amount of protein and a high concentration of K⁺ Neither protein nor lipid become labeled significantly in sieve tubes during short-term translocation experiments with C¹⁴. In general the chemical composition of the assimilate stream is comparable to that of vascular plants and does not, consequently, necessitate a different mechanism for translocation.     

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.     

Analysis of wheat resistance to the cereal aphidSitobion avenae using electrical penetration graphs and flow charts combined with correspondence analysis

The behaviour ofSitobion avenae (F.), was compared on resistant wheat lines ofTriticum monococcum (L.) and a susceptible variety ofTriticum aestivum (L.). Firstly, stylet penetration activities were monitored with the Electrical Penetration Graph (EPG) technique and subsequently analysed using flow charts combined with correspondence analysis. Plant resistance was shown to be associated with repeated penetrations without access to either the xylem or the phloem, and with numerous failures in starting a sustained sap ingestion (as represented by pattern E2). Access to sieve elements of the phloem did not seem to be much affected on resistant plants but it took the aphid three times as long to produce a sap ingestion pattern when maintained on the resistant lineT. monococcum n^o 44 (Tm44) as compared with aphids maintained on susceptible plants. As a result the total time spent in ingesting from sieve elements was reduced by 72% on Tm44. Secondly, direct observations of freely-moving apterous adults were performed. Aphids did not discriminate between resistant and susceptible wheat during the first 30 min of access to test leaves, but only 4 out of 25 aphids were still probing after eight hours on resistant Tm44. The relevance of these results to possible location of the resistance factor(s) are discussed. Although detection of plant resistance before sieve elements are reached can not be rigorously excluded, the factors involved inT. monococcum resistance toS. avenae undoubtedly occur within the phloem vessels.     

TRANSLOCATION IN MACROCYSTIS. III. COMPOSITION OF SIEVE TUBE EXUDATE AND IDENTIFICATION OF THE MAJOR C14-LABELED PRODUCTS1

The major C¹⁴-labeled substance in sieve tube exudate of M. pyrifera is D-mannitol, comprising 3.6% (w/v). No sugars are detectable. Certain amino acids also possess some CWabeling and occur in significantly high concentrations in exudate. The exudate contains negligible ether-soluble lipid, but has a large amount of protein and a high concentration of K⁺. Neither protein nor lipid become labeled significantly in sieve tubes during short-term translocation experiments with C¹⁴. In general the chemical composition of the assimilate stream is comparable to that of vascular plants and does not, consequently, necessitate a different mechanism for translocation.     

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.     

Primitive-like metaphloem sieve elements in the aerial stem ofEquisetum hyemale

Summary Internodal metaphloem sieve elements located near the nodes of aerial stems ofEquisetum hyemale contain very oblique end walls. During maturation, the connections, or plasmodesmata, in these walls undergo little or no structural modification. By contrast, the endwall connections uniting the protoplasts of mature sieve elements elsewhere in the aerial stem ofE. hyemale are pores.This work as supported by U.S. National Science Foundation grant GB 31417 to R. F.Evert.     

Changes in the Endoplasmic Reticulum During Sieve Element Differentiation in Triticum aestivum

The changes in structure of the endoplasmic reticulum (ER) andits associations with other cell components have been studiedin differentiating protophloem sieve elements of root tips ofTriticum aestivum. In the young sieve elements single ER cisternaebearing ribosomes are dispersed in the cytoplasm. As differentiationprogresses ER increases in amount while a small proportion ofit aggregates into stacks or becomes associated with the nuclearenvelope and the mitochondria. These modifications occur inthe last two sieve elements containing ribosomes and coincidewith most dramatic changes in the degenerating nucleus. Stacksconsist of relatively few ER cisternae and may be encounteredfree in the cytoplasm or applied to the nuclear envelope. Electron-densematerial accumulates between the contiguous cisternae of thestacks. ER-attached ribosomes persist even in nearly maturesieve elements, but their pattern of arrangement becomes changed.The structural evidence indicates that only a few highly degradedER elements are retained in fully mature sieve elements. Triticum aestivum, root protophloem, sieve elements, endoplasmic reticulum, differentiation