STUDIES ON THE LEAF OF AMARANTHUS RETROFLEXUS (AMARANTHACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATION IN THE PHLOEM

The vascular system of the leaf of Amaranthus retroflexus L. was examined quantitatively, and plasmolytic studies were carried out on it to determine the solute concentration in cells of the phloem at various locations in the leaf. The proportion of phloem occupied by sieve tubes varies considerably with vein size and leaf size. Collectively, the cross-sectional area of sieve tubes of all tributaries at their points of entry into either a secondary or midvein far exceeds the total cross-sectional area of sieve tubes at the bases of those major veins. In addition, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than total sieve-tube volume of the secondary vein itself. The plasmolytic studies revealed the presence of positive concentration gradients in the sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and from the tip to the base of the midvein. The C₅₀ (the estimated mannitol concentration plasmolyzing, on the average, 50% of the sieve-tube members) was 1.5 m for minor veins and tips of secondary veins and 1.1 m for the bases of secondaries; 1.3 m for the tip of the midvein and 0.6-0.7 m for the midvein in the basal third of the lamina.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.     

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.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATIONS OF THE SIEVE-TUBE MEMBERS

The vascular system of the leaf of Populus deltoides Bartr. ex Marsh, was examined quantitatively, and plasmolytic studies were carried out to determine the solute concentrations of sieve-tube members at various locations in the leaf. Both the total number and total crosssectional area of each cell type decreases with decreasing vein size. Although the proportion of phloem occupied by sieve tubes varies considerably from location to location, a linear relationship exists between cross-sectional area of the vascular bundles and both total and mean cross-sectional area of sieve tubes. Collectively, the cross-sectional area of all tertiary and minor veins feeding into a secondary exceeds the total cross-sectional area of sieve tubes at the base of that secondary. Moreover, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than the total sieve tube volume of the secondary itself. Both tracheary elements and sieve-tube members undergo a reduction in both total and mean crosssectional area in the constricted zone at the base of the leaf. The plasmolytic studies revealed the presence of positive concentration gradients in sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and their associated subjacent midvein bundles and from the upper to lower portions of the median bundle of the midvein.     

THE TIME COURSE OF SIEVE TUBE AND VESSEL REGENERATION AND THEIR RELATION TO PHLOEM ANASTOMOSES IN MATURE INTERNODES OF COLEUS

Quantitative counts of regenerative sieve tubes and vessels were made in a large number of samples of mature internode #5 of C. blumei, with concomitant study of the fine details of vascular regeneration and the occurrence of the normally developing phloem anastomoses. Such anastomoses were found in many of the plants, but their average number in the small regenerating area was low (viz., 0.9 ± 0.2). With the phloem anastomoses excluded from the counts, the time course of regeneration was clear cut—no strands completed their regeneration around the wound until three days after wounding. More regenerative sieve tubes completed their differentiation under all conditions than did regenerative vessels. The number of sieve tubes and vessels regenerated by four days was closely related to the number of preexisting bundles of that type of vascular cell that had been severed by the transverse wound. The ratio of bundles severed by the wound in the phloem to those in the xylem was 2.14, and the ratio of the regenerative sieve tubes to the regenerative vessels was 2.24. For both tracheary and sieve tube cells the initial regeneration was strongly polar (mostly above the wound), as expected from earlier IAA transport data. The path of tracheary regeneration was obviously related to that of the sieve tubes on the other side of the cambium.     

VASCULAR SYSTEM OF LODICULES OF DACTYLIS GLOMERATA L.

The vascular system for the two lodicules in a floret of Dactylis glomerata L. was studied in serial sections. The floret stele contained a few modified tracheary elements and xylem transfer cells enveloped by a phloem of squat sieve-tube members and intermediary cells. A single sieve tube and associated phloem parenchyma exited the right and left sides of the stele and upon nearing the base of each lodicule branched and formed the minor veins of the lodicule. The minor veins underwent limited branching and anastomosing to form a small three-dimensional system which described an arc during its ascent in the adaxial portion of each lodicule. The sieve tubes in the minor veins extended halfway up the lodicule and contained short sieve-tube members with transverse, slightly oblique, or lateral simple sieve plates. The associated phloem parenchyma cells were intermediary cells, companion cells, and less intimate parenchyma cells. Intermediary cells terminated the minor veins and touched the distal ends of the terminal sieve-tube members, which lacked distal sieve plates. Although the transverse area of the sieve-tube members remained constant up the lodicule, the transverse area of the associated phloem parenchyma fluctuated.     

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade‐off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier–Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.     

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.     

Auxin promotes dormancy callose removal from the phloem ofMagnolia kobus and callose accumulation and earlywood vessel differentiation inQuercus robur

During winter, the phloem of the diffuse-porous tree magnolia (Magnolia kobus DC.) is dormant and is characterized by heavy deposits of dormancy callose. Application of 1-naphthaleneacetic acid (NAA) to either the top or the lower ends of excised dormant branches before bud break resulted in the removal of the dormancy callose from the sieve tubes. In both intact and auxintreated branches, callose degradation occurred first in the recently formed sieve tubes. There was no new vessel differentiation in magnolia before bud break. In contrast, the sieve tubes of the ring-porous oak (Quercus robur L.), which possess massive dormancy callose deposits during winter, were almost callose-free just before bud break. Application of auxin to the top of excised branches before bud break resulted in callose accumulation on the most recently formed sieve tubes. The first earlywood vessels were evident in oak before bud break, and their numbers were increased by auxin application. The early development of phloem and xylem (before bud break) in ring-porous species is an ecological adaptation which prepares the vascular system of these trees to function immediately at the beginning of their growing season which is relatively short.     

Translocation and accumulation of translocate in the sugar beet petiole

Accumulation of translocate during steady-state labeling of photosynthate was measured in the source leaf petioles of sugar beet (Beta vulgaris L. monogerm hybrid). During an 8-hr period, 2.7% of the translocate or 0.38 μg carbon/min was accumulated per cm petiole. Material was stored mainly as sucrose and as compounds insoluble in 80% ethanol. The minimum peak velocity of translocation approached an average of 54 cm/hr as the specific activity of the ¹⁴CO₂ pulse was progressively increased. The ratio of cross sectional area required for translocation to actual sieve tube area in the petiole was 1.2. A regression analysis of translocation rate versus sieve tube cross sectional area yielded a coefficient of 0.76. The specific mass transfer rate in the petiole was 1.4 g/hr cm² phloem or 4.8 g/hr cm² sieve tube. Histoautoradiographic studies indicated that translocation occurs through the area of phloem occupied by sieve tubes and companion cells while storage occurs in these cells plus cambium and phloem parenchyma cells. The ability of the petiole to act as a sink for translocate is consistent with the concept that storage along path tissue serves to buffer sucrose concentration in the translocate during periods of fluctuating assimilation.     

The proportion of sieve elements in the phloem of some tropical trees

Summary Measurements have been made of the proportion of the area of sieve elements in the cross-sectional area of the secondary phloem of trees of two tropical genera in which the presence of storied sieve plates makes the recognition of sieve elements particularly easy. This proportion, often accepted as one fifth in the literature on phloem and translocation, rises as high as three quarters in one of the trees measured.It is with regret that we record the death of Dr. Lawton in the closing stages of this investigation.     

Sink Effect of Cytokinin in Detached Leaves Is Not Caused by Structural Rearrangements of Phloem

The sink effect of cytokinin is manifested as a decrease in source capacity and the induction of sink activity in the phytohormone-treated region of a mature excised leaf. In order to find out whether this effect was due to the direct action of cytokinin on the phloem structure, two types of phloem terminals were examined. In pumpkin (Cucurbita pepo L.) leaves, the phloem terminals are open; i.e., they are linked to mesophyll by numerous symplastic connections, which are located in narrow areas called plasmodesmal pit fields. In broad bean (Vicia faba L.) leaves, the phloem terminals belong to the closed type and have no symplastic links with mesophyll. The electron microscopic study of terminal phloem did not reveal any structural changes in the companion cells, which could account for the suppression of assimilate export. The treatment of leaves with cytokinin neither disturbed the structure of plasmodesmal pit fields in pumpkin leaves nor eliminated the wall protuberances (the ingrowths promoting phloem loading) in bean leaves. No evidence was obtained that the cytokinin-induced import of assimilates in mature leaves is caused by the recovery of meristematic activity, i.e., by either formation of new phloem terminals having immature sieve elements capable of unloading or by the development of new sieve elements within the existing veins. Cytokinin did not induce de novo formation of phloem elements. Structural characteristics of the leaf phloem, such as the number of branching orders in the venation pattern, the number of vein endings per areole, the number of areoles per leaf, the area of one areole, and the number of sieve elements per bundle remained unaltered. It is concluded that the sink effect of cytokinin in excised leaves cannot be determined by alteration of the phloem structure.     

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.     

Sieve-element differentiation and fluoresceine translocation in wound-phloem of pea roots after complete severance of the stele

Experimental interruption of the root stele of Pisum sativum L. induces in the cortex tissue the development of wound-sieve tubes which bridge the wound and reconnect the vascular stumps. Outside the stele, sieve plates arise from primary pit fields. This origin is confirmed by the distribution of future sieve pores over the original parenchyma cell wall and by remnants of the pitfield cavity in developing sieve plates. Differentiation of wound-sieve elements is similar to that of bundle-sieve elements and includes the chromatolytic disintegration of nuclei as well as the development of typical sieve pores arising from pit-field plasmodesmata. The completion of first woundsieve tubes (indicated by a continuous chain of anilin-blue-positive sieve plates by-passing the wound) was observed 55–62 h after wounding. However, effective translocation, visualized with fluoresceine as a phloem-mobile marker, was not found until 10 h (on average) later. It is suggested that this time delay corresponds to the maturing of the last link within a chain of wound-sieve-tube members. Presumably, enucleate sieve elements with widened pores are a prerequisite for effective phloem translocation.Abbreviations DAPI 4,6-diamidino-2-phenylindole·2 H₂O - ER endoplasmic reticulum Preliminary results of this investigation have been presented at the International Phloem Transport Conference in Asilomar, Cal., USA 1985 (cf. Schulz 1986c)     

Localization of the beet mild yellowing virus inSinapis alba L.

Virus particles of isometric shape with a diameter of 26 nm were found in the sieve tubes and accompanying phloem cells in ultrathin sections prepared from the nerves of white mustard (Sinapis alba L.) leaves and roots infected with the beet mild yellowing virus (BMYV). BMYV particles were much more frequent in the roots ofSinapis alba plants. Isometric particles were not found in the leaves and roots of healthy mustard plants.     

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.     

Structurally,phloem unloading in the maize leaf cannot be symplastic

Developing longitudinal vascular bundles of the leaf blades of maize (Zea mays L. cv. W273) were examined with the transmission electron microscope to determine the frequency of plasmodesmata between the sieve tubes and their neighboring cells. Of particular interest were the protophloem sieve tubes, the first sieve tubes to mature in importing (all large and some intermediate) bundles. The protophloem sieve tubes, most of which lack companion cells, intergrade structurally with the thin-walled metaphloem sieve tubes. Both the protophloem sieve tubes and the thin-walled metaphloem sieve tubes and their companion cells (the sieve tube-companion cell complexes) are virtually isolated symplastically from the rest of the leaf, precluding a symplastic mechanism of phloem unloading in the leaf blade of maize.     

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

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

Association between photosynthesis and contrasting features of minor veins in leaves of summer annuals loading phloem via symplastic versus apoplastic routes

Foliar vascular anatomy and photosynthesis were evaluated for a number of summer annual species that either load sugars into the phloem via a symplastic route (Cucumis sativus L. cv. Straight Eight; Cucurbita pepo L. cv. Italian Zucchini Romanesco; Citrullus lanatus L. cv. Faerie Hybrid; Cucurbita pepo L. cv. Autumn Gold) or an apoplastic route (Nicotiana tabacum L.; Solanum lycopersicum L. cv. Brandywine; Gossypium hirsutum L.; Helianthus annuus L. cv. Soraya), as well as winter annual apoplastic loaders (Spinacia oleracea L. cv. Giant Nobel; Arabidopsis thaliana (L.) Heynhold Col‐0, Swedish and Italian ecotypes). For all summer annuals, minor vein cross‐sectional xylem area and tracheid number as well as the ratio of phloem loading cells to phloem sieve elements, each when normalized for foliar vein density (VD), was correlated with photosynthesis. These links presumably reflect (1) the xylem's role in providing water to meet foliar transpirational demand supporting photosynthesis and (2) the importance of the driving force of phloem loading as well as the cross‐sectional area for phloem sap flux to match foliar photosynthate production. While photosynthesis correlated with the product of VD and cross‐sectional phloem cell area among symplastic loaders, photosynthesis correlated with the product of VD and phloem cell number per vein among summer annual apoplastic loaders. Phloem cell size has thus apparently been a target of selection among symplastic loaders (where loading depends on enzyme concentration within loading cells) versus phloem cell number among apoplastic loaders (where loading depends on membrane transporter numbers).     

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.